Food Materials Comprising Filamentous Fungal Particles and Membrane Bioreactor Design

ABSTRACT

Methods of production of edible filamentous fungal biomat formulations are provided as standalone protein sources and/or protein ingredients in foodstuffs as well as a one-time use or repeated use self-contained biomat reactor comprising a container with at least one compartment and placed within the compartment(s), a feedstock, a fungal inoculum, a gas-permeable membrane, and optionally a liquid nutrient medium.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalPatent Application 62/811,421, filed 27 Feb. 2019, the entirety of whichis incorporated herein by reference.

TECHNICAL FIELD

This application relates to edible filamentous fungi and providesmethods of preparing edible fungi for use in foodstuffs, liquid andsolid formulations of edible fungi, as well as uses and methodsassociated therewith, foodstuffs containing edible filamentous fungi,and methods and uses thereof.

BACKGROUND

The United Nations listed the world population as 7.5 billion in August2017 and predicts that figure to grow to 8 billion in 2023 and to be 10billion in 2056. In a related report, the Food and AgriculturalOrganization of the United Nations (FAO) estimates that if the globalpopulation reaches 9.1 billion by 2050, world food production will needto rise by 70% and to double in the developing world. That increase infood production will need to occur despite rising energy costs,decreasing underground aquifer resources, loss of farmland to urbansprawl, and increasingly severe weather due to climate change (e.g.increased temperatures, increased drought, increased flooding, etc.).This is a particular challenge for countries such as Africa which,according to 2009 figures, already has inadequate protein intake andcountries such as China, India, Pakistan, and Indonesia which are atrisk of inadequate protein intake. In addition, the global demand isforecasted for 2040 to increase by 60% for meat and 50% for dairy.

But not all protein sources are created equal. Animal-based foods (meat,eggs, dairy) provide “complete” proteins as they contain all of theessential amino acids; that is, methionine, leucine, isoleucine,phenylalanine, valine, threonine, histidine, tryptophan and lysine.Plant-based foods, while containing some essential amino acids,generally lack the complete set. For example, the protein found instarchy roots lacks the essential amino acid lysine, which must then beobtained from another food in the diet. Beans and legumes contain highlevels of lysine, but they lack the essential amino acid methionine.Although it is possible to build a complete protein by pairing plantfoods, ensuring a nutritionally balanced diet is much easier withcomplete proteins.

One non-animal source of complete protein is obtained from ediblefilamentous fungi, such as Fusarium venenatum (formerly classified andFusarium graminearum). However, to date protein production from thesesources has required significant investment in energy resources andproduction equipment, such as capital-intensive bioreactors andcentrifuges. There remains a need for growth, harvesting, and foodstuffproduction methods that require low energy, consume few naturalresources, and are low cost. The current invention solves theseproblems.

In addition, one area of reducing the logistics supply associated withresponding to natural disasters, logistically isolated environments ormilitary and/or space/extraterrestrial missions is the closure of lifesupport loops, particularly waste streams, while providing missioncritical products such as nutritional and appetizing foods, fuels,metabolite expression platforms, building materials and/or microbialfactories. Oftentimes these types of environments have no or limitedaccess to sterile facilities and/or require a sealed aseptic system tofully contain the waste stream and/or food, fuel and materials produced.For example, work by the European Space Agency (Expeditions 25-28,Growth and Survival of Colored Fungi in Space (CFS-A)) demonstrated thatfungi can grow inside the space station and could decompose food andother organic materials in humid conditions; here containment of thefungal system is paramount to preventing inadvertent contamination ofother supplies and surfaces. In addition to the need to decompose foodand waste in the developing area of space travel, these needs are alsopresent when dealing with natural disasters, in-theater militaryoperations, wilderness operations, situations in the third world wheresanitation and refrigeration are not reliable, confined spaces,logistically difficult arenas and in some agricultural/industrialoperations. Having a self-contained aseptic system that operatesefficiently with a minimum of space, energy, and maintenance is needed.

A robust and efficient portable self-contained biomat reactor systemthat is able to convert a wide variety of waste streams into a multitudeof valuable products addresses these problems. The current disclosuredescribes a simple aseptic bioreactor platform that requires noagitation, no active aeration, no external energy source duringfermentation (other than temperature control), generates minimal to nowaste residues, requires little water, and produces dense, easilyharvested, textured biomats. In addition, the self-contained biomatreactor system can be portable and/or scalable for larger, moreconcentrated missions and/or populations.

SUMMARY

It is one aspect of the present invention to provide a food material,comprising particles of a filamentous fungus belonging to an orderselected from the group consisting of Mucorales Ustilaginales,Russulales, Polyporales, Agaricales, Pezizales and Hypocreales, whereinthe filamentous fungus comprises greater than about 40 wt. % proteincontent and less than about 8 wt. % RNA content.

In embodiments, the filamentous fungus may belong to a family selectedfrom the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae,Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae,Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae,Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, andCordycipitaceae.

In embodiments, the filamentous fungus may belong to a species selectedfrom the group consisting of Rhizopus oligosporus, Ustilago esculenta,Hericululm erinaceus, Polyporous squamosus, Grifola fondrosa, Hypsizygusmarmoreus, Hypsizygus ulmarius (elm oyster) Calocybe gambosa, Pholiotanameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata,Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (pearl),Pleurotus ostreatus var. columbinus (Blue oyster), Tuber borchii,Morchella esculenta, Morchella conica, Morchella importuna, Sparassiscrispa (cauliflower), Fusarium venenatum, strain MK7 (ATCC AccessionDeposit No. PTA-10698), Disciotis venosa, Clonostachys rosea, Cordycepsmilitaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes,Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, andLeucoagaricus spp.

In embodiments, the filamentous fungus may be a Fusarium species.

In embodiments, the filamentous fungus may be Fusarium venenatum.

In embodiments, the filamentous fungus may be strain MK7 (ATCC AccessionDeposit No. PTA-10698).

In embodiments, the filamentous fungus may have a characteristicselected from the group consisting of (a) comprising greater than about45 wt. % protein content; (b) comprising greater than about 50 wt. %protein content; (c) comprising greater than about 55 wt. % proteincontent; (d) comprising greater than about 60 wt. % protein content; (e)comprising less than about 5 wt. % RNA content; (f) comprising less thanabout 4 wt. % RNA content; (g) comprising less than about 3 wt. % RNAcontent; (h) comprising less than about 2 wt. % RNA content; andcombinations of one of a-d and one of e-h.

In embodiments, the filamentous fungus may comprise less than about 10ppm of a mycotoxin selected from the group consisting of Alfatoxin B1,Alfatoxin B2, Alfatoxin G1, Alfatoxin G2, Fumonisin B1, Fumonisin B2,Fumonisin B3, Ochratoxin A, Nivalenol, Deoxynivalenol, Acetyldeoxynivalenol, Fusarenon X, T-2 Toxin, HT-2 Toxin, Neosolaniol,Diacetoxyscirpenol zearalenone, beauvericin, fusarin C, fusaric acid,and any combinations thereof.

The filamentous fungus may comprise less than about 10 ppm, or less thanabout 9 ppm, or less than about 8 ppm, or less than about 7 ppm, or lessthan about 6 ppm, or less than about 5 ppm, or less than about 4 ppm, orless than about 3 ppm, or less than about 2 ppm, or less than about 1ppm, or less than about 0.9 ppm, or less than about 0.8 ppm, or lessthan about 0.7 ppm, or less than about 0.6 ppm of the selectedmycotoxin, or alternatively less than about any tenth of a part permillion equal to or less than 10 ppm. In particular embodiments, theselected mycotoxin may be a fumonisin or combination of fumonisins,beauvericin, fusarin C, fusaric acid, and combinations thereof.

In embodiments, the filamentous fungus may comprise less than about 10ppm total mycotoxin content, or less than about 9 ppm total mycotoxincontent, or less than about 8 ppm total mycotoxin content, or less thanabout 7 ppm total mycotoxin content, or less than about 6 ppm totalmycotoxin content, or less than about 5 ppm total mycotoxin content, orless than about 4 ppm total mycotoxin content, or less than about 3 ppmtotal mycotoxin content, or less than about 2 ppm total mycotoxincontent, or less than about 1 ppm total mycotoxin content, or less thanabout 0.9 ppm total mycotoxin content, or less than about 0.8 ppm totalmycotoxin content, or less than about 0.7 ppm total mycotoxin content,or less than about 0.6 ppm total mycotoxin content, or alternativelyless than about any tenth of a part per million equal to or less than 10ppm total mycotoxin content.

In embodiments, the filamentous fungus may comprise greater than about15 wt. % of branched chain amino acids.

In embodiments, the particles of filamentous fungus may be in the formof a flour. The flour may, but need not, have a particle size of from30-400 microns. The flour may, but need not, have a particle size of nomore than about 400 microns, no more than about 390 microns, no morethan about 380 microns, no more than about 370 microns, no more thanabout 360 microns, no more than about 350 microns, no more than about340 microns, no more than about 330 microns, no more than about 320microns, no more than about 310 microns, no more than about 300 microns,no more than about 290 microns, no more than about 280 microns, no morethan about 270 microns, no more than about 260 microns, no more thanabout 250 microns, no more than about 240 microns, no more than about230 microns, no more than about 220 microns, no more than about 210microns, no more than about 200 microns, no more than about 190 microns,no more than about 180 microns, no more than about 170 microns, no morethan about 160 microns, no more than about 150 microns, no more thanabout 140 microns, no more than about 130 microns, no more than about120 microns, no more than about 110 microns, no more than about 100microns, no more than about 90 microns, no more than about 80 microns,no more than about 70 microns, no more than about 60 microns, no morethan about 50 microns, no more than about 40 microns, no more than about30 microns, no more than about 20 microns, no more than about 10microns, no more than about 9 microns, no more than about 8 microns, nomore than about 7 microns, no more than about 6 microns, no more thanabout 5 microns, no more than about 4 microns, no more than about 3microns, no more than about 2 microns, or no more than about 1 micron,or alternatively no more than about any whole number of microns betweenabout 1 micron and about 400 microns. The particle size may be any oneor more of a D₁₀ particle size, a D₂₅ particle size, a D₅₀ particlesize, a D₇₅ particle size, a D₉₀ particle size, or a weight-averageparticle size. In some embodiments, substantially all particles may havea particle size of at least about 30 microns and no more than about 400microns.

In embodiments, the particles may have a particle length of about 0.05mm to about 500 mm, a particle width of about 0.03 mm to about 7 mm, anda particle height of about 0.03 mm to about 1.0 mm. The particles may,but need not, have a particle length, a particle width, and a particleheight that are all more than about 0.02 mm, more than about 0.03 mm,more than about 0.04 mm, more than about 0.05 mm, more than about 0.06mm, more than about 0.07 mm, more than about 0.08 mm, more than about0.09 mm, more than about 0.10 mm, more than about 0.11 mm, more thanabout 0.12 mm, more than about 0.13 mm, more than about 0.14 mm, morethan about 0.15 mm, more than about 0.16 mm, more than about 0.17 mm,more than about 0.18 mm, more than about 0.19 mm, or more than about0.20 mm, or alternatively more than about any whole number of micronsthat is at least about 20 microns.

In embodiments, the food material may be a liquid dispersion of theparticles of filamentous fungus. The liquid dispersion may, but neednot, be produced under nitrogen. The liquid dispersion of the particlesof filamentous fungus may, but need not, be stable for at least about 1day.

In embodiments, the food material may be vegan.

In embodiments, the particles of filamentous fungus may be the soleprotein component present in the food material.

In embodiments, the particles of filamentous fungus may comprise allessential amino acids. The particles of filamentous fungus may, but neednot, comprise at least one branched-chain amino acid selected from thegroup consisting of leucine, isoleucine, and valine.

In embodiments, the filamentous fungal particles may be nonviable.

It is another aspect of the present invention to provide a yogurt analogfood product comprising particles of a filamentous fungus belonging toan order selected from the group consisting of Mucorales, Ustilaginales,Russulales, Polyporales, Agaricales, Pezizales and Hypocreales, whereinthe filamentous fungus comprises greater than about 40 wt. % proteincontent and less than about 8 wt. % RNA content.

In embodiments, the filamentous fungus may belong to a family selectedfrom the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae,Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae,Agaricaceae, Pleurotaceae, Tuberaceae, Morchellaceae, Sparassidaceae,Physalacriaceae, Omphalotaceae, Nectriaceae, Bionectriaceae, andCordycipitaceae.

In embodiments, the filamentous fungus may belong to a species selectedfrom the group consisting of Rhizopus oligosporus, Ustilago esculenta,Hericululm erinaceus, Polyporous squamosus, Grifola fondrosa, Hypsizygusmarmoreus, Hypsizygus ulmarius (elm oyster) Calocybe gambosa, Pholiotanameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata,Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (pearl),Pleurotus ostreatus var. columbinus (Blue oyster), Tuber borchii,Morchella esculenta, Morchella conica, Morchella importuna, Sparassiscrispa (cauliflower), Fusarium venenatum, strain MK7 (ATCC AccessionDeposit No. PTA-10698), Disciotis venosa, Clonostachys rosea, Cordycepsmilitaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes,Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, andLeucoagaricus spp.

In embodiments, the filamentous fungus may be a Fusarium species.

In embodiments, the filamentous fungus may be Fusarium venenatum.

In embodiments, the filamentous fungus may be strain MK7 (ATCC AccessionDeposit No. PTA-10698).

In embodiments, the filamentous fungus may have a characteristicselected from the group consisting of (a) comprising greater than about45 wt. % protein content; (b) comprising greater than about 50 wt. %protein content; (c) comprising greater than about 55 wt. % proteincontent; (d) comprising greater than about 60 wt. % protein content; (e)comprising less than about 5 wt. % RNA content; (f) comprising less thanabout 4 wt. % RNA content; (g) comprising less than about 3 wt. % RNAcontent; (g) comprising less than about 2 wt. % RNA content; andcombinations of one of a-d and one of e-h.

In embodiments, the ratio of the filamentous fungal particles to watermay range from about 1:10 to about 10:1.

In embodiments, the ratio of the filamentous fungal particles to watermay be selected from the group consisting of about 1:3, about 1:2, about1:1 and about 2:1.

In embodiments, the yogurt analog food product may further comprise aninvert sugar.

In embodiments, the yogurt analog food product may further comprise athickening agent.

In embodiments, cells of the filamentous fungus may be lysed.

In embodiments, the yogurt analog food product may further compriseLactobacillus bulgaricus and Streptococcus thermophilus.

In embodiments, the product may be vegan.

In embodiments, the filamentous fungal particles may comprise allessential amino acids.

In embodiments, the filamentous fungal particles may be the sole proteincomponent.

In embodiments, the filamentous fungal particles may be nonviable.

In embodiments, the yogurt analog food product may further comprise arennet. The rennet may, but need not, be from a source selected from thegroup consisting of an animal source, a vegetarian source and amicrobial source. The rennet may, but need not, be from a sourceselected from the group consisting of a vegetarian source and amicrobial source.

In embodiments, the product may be free of milk solids.

In embodiments, the yogurt analog food product may further comprise aprobiotic.

In embodiments, the yogurt analog food product may further comprise anenzymatic water.

It is another aspect of the present invention to provide a foammaterial, comprising particles of a filamentous fungal biomat; and aliquid phase, wherein the solids content of the foam material is betweenabout 5% and about 30%, and wherein the foam is stable. The liquid phasemay, but need not, be an aqueous phase, i.e. comprise water.

In embodiments, the foam material may not collapse spontaneouslyimmediately upon cessation of the foaming process during its production.

In embodiments, the foam material may be stable for at least about 7days.

In embodiments, the foam material may have an overrun of at least about10%.

In embodiments, the filamentous fungal biomat may comprise a Fusariumspecies.

In embodiments, the foam material may be free of milk solids.

It is another aspect of the present invention to provide a food productcomprising the foam material.

It is another aspect of the present invention to provide a bioreactor,comprising a container; at least one membrane disposed within or on asurface of the container, the at least one membrane comprising a firstsurface and a second surface; a feedstock for the growth of afilamentous fungus, contacting the first surface of the at least onemembrane; and a filamentous fungus inoculum, disposed on either thefirst surface or the second surface of the at least one membrane,wherein, upon culturing the inoculum in the bioreactor, a biomat of thefilamentous fungus forms on the second surface of the at least onemembrane after a biomat growth period. As used herein, the term“membrane,” unless otherwise specified, refers to any flexible orsemi-flexible enclosing or separating part that forms a plane or filmand separates two environments, and that has pores therethrough enablingexchange of at least a portion of a fluid between the two environments.

In embodiments, the container may be a bag, wherein the first and secondsurfaces of the at least one membrane are first and second surfaces ofat least a portion of the bag.

In embodiments, the feedstock may be subjected to a positive or negativepressure imparted on a side of the feedstock opposite at least one ofthe first surface and the second surface of the at least one membrane.

In embodiments, the bioreactor may further comprise cyanobacteria,wherein the cyanobacteria provide at least one of oxygen gas and carbonto promote the growth of the biomat.

In embodiments, at least one of the following may be true: i) a densityof the biomat is at least about 0.05 grams per cubic centimeter; and ii)a density of the biomat after drying is at least about 0.01 grams percubic centimeter.

In embodiments, the biomat may comprise at least one layer.

In embodiments, the biomat may have a tensile strength of at least about3 kilopascals or at least about 30 grams-force per square centimeter.The biomat may, but need not, have a tensile strength of at least about100 kilopascals or at least about 1,020 grams-force per squarecentimeter.

In embodiments, the at least one membrane may comprise at least onepolymer selected from the group consisting of polypropylenes,polytetrafluoroethylenes, polycarbonates, polyamides, cellulose acetate,polyvinylidene fluorides, mixed cellulose esters, polyethersulfones,polyethylenes, and polypyrroles.

In embodiments, the at least one membrane may comprise at least onematerial selected from the group consisting of polypropylene fabrics,polytetrafluoroethylene fabrics, and a nylon net filter.

In embodiments, the at least one membrane may comprise at least one of aglass fiber material and a porous ceramic material.

In embodiments, an average pore size of the at least one membrane may bebetween about 0.2 μm and about 25 μm. The average pore size of the atleast one membrane may, but need not, be between about 5 μm and about 11μm.

In embodiments, the container may be enclosed and substantiallyairtight, wherein the container encloses a gas headspace into which thebiomat grows.

In embodiments, the biomat may separate from the at least one membranespontaneously.

In embodiments, when the biomat is removed from the at least onemembrane, a new inoculum of filamentous fungi may remain on the at leastone membrane.

In embodiments, the filamentous fungus may belong to an order selectedfrom the group consisting of Mucorales, Ustilaginales, Russulales,Polyporales, Agaricales, Pezizales, and Hypocreales.

In embodiments, the filamentous fungus may belong to a family selectedfrom the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae,Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae,Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae,Ophiocordycipitaceae, Tuberaceae, Morchellaceae, and Cordycipitaceae.

In embodiments, the filamentous fungus may be selected from the groupconsisting of strain MK7 (ATCC Accession Deposit No. PTA-10698),Fusarium venenatum, Rhizopus oligosporus, Ustilago esculenta, Hericulumerinaceus, Polyporous squamosus, Grifola fondrosa, Hypsizygus marmoreus,Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus,Stropharia rugosoannulata, Hypholoma lateritium, Pluerotus eryngii,Tuber borchii, Morchella esculenta, Morchella conica, Disciotis venosa,Ophiocordyceps sinensis and Cordyceps militaris, Trametes versicolor,Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotusdjamor, Pleurotus ostreatus, Leucoagaricus holosericeus, Calvatiafragilis, Handkea utriformis, and Pholiota adiposa.

In embodiments, the feedstock may comprise at least one of feces of ananimal and urine of an animal. The animal may, but need not, be a human.

In embodiments, the at least one membrane may be a single compositemembrane, wherein the first surface comprises a first material and thesecond surface comprises a second material.

In embodiments, the at least one membrane may comprise at least a firstmembrane and a second membrane, wherein the first surface is a surfaceof the first membrane and the second surface is a surface of the secondmembrane. The first and second membranes may, but need not, be inphysical contact with each other.

In embodiments, the bioreactor may further comprise a selectivegas-permeable membrane, wherein a first gas produced during growth ofthe biomat is selectively separated into a gas headspace on a first sideof the selective gas-permeable membrane. A second gas produced duringgrowth of the biomat may, but need not, be selectively separated into agas headspace on a second side of the membrane. In some embodiments, agas dissolved or dispersed in a liquid feedstock, or otherwise disposedon a feedstock side of the membrane, may be selectively separated fromthe feedstock and passed to an opposing side of a membrane.

It is another aspect of the present invention to provide a method forproducing a biomat of a filamentous fungus, comprising inoculating afilamentous fungus in a bioreactor, wherein the bioreactor comprises acontainer; at least one membrane disposed within or on a surface of thecontainer, the at least one membrane comprising a first surface and asecond surface, wherein either or both of the first and second surfacesare adapted to receive thereon the inoculum of the filamentous fungus;and a feedstock for the growth of a filamentous fungus, contacting thefirst surface of the at least one membrane.

In embodiments, the container may be a bag, wherein the first and secondsurfaces of the at least one membrane are first and second surfaces ofat least a portion of the bag.

In embodiments, the feedstock may be subjected to a positive or negativepressure imparted on a side of the feedstock opposite the first surfaceof the at least one membrane. The positive or negative pressure may, butneed not, facilitate the inoculating step.

In embodiments, the method may further comprise providing cyanobacteriain the bioreactor, wherein the cyanobacteria provide at least one ofoxygen gas and carbon to promote the growth of the biomat.

In embodiments, at least one of the following may be true: i) a densityof the biomat after harvesting is at least about 0.6 grams per cubiccentimeter; and ii) a density of the biomat after harvesting and dryingis at least about 0.1 grams per cubic centimeter.

In embodiments, the biomat may comprise at least one layer.

In embodiments, during or after the harvesting step, the biomat may havea tensile strength of at least about 3 kilopascals or at least about 30grams-force per square centimeter. During or after the harvesting step,the biomat may, but need not, have a tensile strength of at least about100 kilopascals or at least about 1,020 grams-force per squarecentimeter.

In embodiments, the at least one membrane may comprise at least onepolymer selected from the group consisting of polypropylenes,polytetrafluoroethylenes, polycarbonates, polyamides, cellulose acetate,polyvinylidene fluorides, mixed cellulose esters, polyethersulfones,polyethylenes, and polypyrroles.

In embodiments, the at least one membrane may comprise at least onematerial selected from the group consisting of polypropylene fabrics,polytetrafluoroethylene fabrics, and a nylon net filter.

In embodiments, the membrane may comprise at least one of a glass fibermaterial and a porous ceramic material.

In embodiments, an average pore size of the at least one membrane may bebetween about 0.2 μm and about 25 μm. An average pore size of the atleast one membrane may, but need not, be between about 5 μm and about 11μm.

In embodiments, the container may be enclosed and substantiallyairtight, wherein the container encloses a gas headspace into which thebiomat grows.

In embodiments, the biomat may separate from the at least one membranespontaneously.

In embodiments, the method may further comprise harvesting the biomat,wherein, when the biomat is removed from the at least one membrane, anew inoculum of filamentous fungi remains on the at least one membrane.

In embodiments, the filamentous fungus may belong to an order selectedfrom the group consisting of Mucorales, Ustilaginales, Russulales,Polyporales, Agaricales, Pezizales, and Hypocreales.

In embodiments, the filamentous fungus may belong to a family selectedfrom the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae,Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae,Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae,Morchellaceae, and Cordycipitaceae.

In embodiments, the filamentous fungus may be selected from the groupconsisting of strain MK7 (ATCC Accession Deposit No. PTA-10698),Fusarium venenatum, Rhizopus oligosporus, Ustilago esculenta, Hericulumerinaceus, Polyporous squamosus, Grifola fondrosa, Hypsizygus marmoreus,Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus,Stropharia rugosoannulata, Hypholoma lateritium, Pluerotus eryngii,Tuber borchii, Morchella esculenta, Morchella conica, Disciotis venosa,Ophiocordyceps sinensis and Cordyceps militaris.

In embodiments, the feedstock may comprise at least one of feces of ananimal and urine of an animal. The animal may, but need not, be a human.

In embodiments, the at least one membrane may be a single compositemembrane, wherein the first surface comprises a first material and thesecond surface comprises a second material.

In embodiments, the at least one membrane may comprise at least a firstmembrane and a second membrane, wherein the first surface is a surfaceof the first membrane and the second surface is a surface of the secondmembrane. The first and second membranes may, but need not, be inphysical contact with each other.

In embodiments, the bioreactor may further comprise a selectivegas-permeable membrane, wherein a first gas produced during growth ofthe biomat is selectively separated into a gas headspace on a first sideof the selective gas-permeable membrane. A second gas produced duringgrowth of the biomat may, but need not, be selectively separated into agas headspace on a second side of the membrane.

It is another aspect of the present invention to provide a method forproducing fresh water, comprising inoculating a filamentous fungus in abioreactor, wherein the bioreactor comprises a container; and afeedstock for the growth of a filamentous fungus; culturing thefilamentous fungus to form a biomat on at least one of a surface of thefeedstock and a surface of a membrane of the bioreactor, wherein thefilamentous fungus produces water as a byproduct during formation orgrowth of the biomat; and collecting water produced by the formation orgrowth of the biomat.

In embodiments, the feedstock may comprise at least one of the feces ofan animal and the urine of an animal. The animal may, but need not, be ahuman.

In embodiments, the method may further comprise recycling the collectedwater to the bioreactor.

In embodiments, the method may further comprise preparing a feedstockcomprising the collected water. The method may, but need not, furthercomprise recycling the prepared feedstock comprising the collected waterto the bioreactor.

It is another aspect of the present invention to provide a method forproducing a gas, comprising inoculating a filamentous fungus in abioreactor, wherein the bioreactor comprises a container; and afeedstock for the growth of a filamentous fungus; culturing thefilamentous fungus to form a biomat on at least one of a surface of thefeedstock and a surface of a membrane of the bioreactor, wherein thefilamentous fungus produces the gas as a metabolic byproduct duringgrowth of the biomat; and collecting the gas produced by the growth ofthe biomat.

In embodiments, the gas may be selected from the group consisting ofammonia, an ammonium species, hydrogen gas, and a volatile ester.

It is one aspect of the present invention to provide a method forproducing a biomat of a filamentous fungus, comprising (a) inoculatingan effective amount of cells of at least one filamentous fungus to afirst aliquot of growth medium to produce an inoculated growth medium;(b) incubating the inoculated growth medium for a first time to producean initial biomat; (c) removing at least a portion of the first aliquotof growth medium and adding a second aliquot of growth medium to providea refreshed growth medium; and (d) incubating the refreshed growthmedium for a second time to produce a finished biomat.

In embodiments, the dry-mass density of the finished biomat may be atleast about 75 grams per liter.

In embodiments, the biomat may comprise greater than about 40 wt %protein and less than about 8 wt % RNA.

In embodiments, the at least one filamentous fungus may belong to anorder selected from the group consisting of Ustilaginales, Russulales,Polyporales, Agaricales, Pezizales, and Hypocreales.

In embodiments, the at least one filamentous fungus may belong to afamily selected from the group consisting of Ustilaginaceae,Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae,Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae,Omphalotaceae, Tuberaceae, Morchellaceae, and Cordycipitaceae

In embodiments, the at least one filamentous fungus is selected from thegroup consisting of strain MK7 (ATCC Accession Deposit No. PTA-10698),Fusarium venenatum, Ustilago esculenta, Hericulum erinaceus, Polyporoussquamosus, Grifola fondrosa, Hypsizygus marmoreus, Calocybe gambosa,Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Strophariarugosoannulata, Hypholoma lateritium, Pluerotus eryngii, Tuber borchii,Morchella esculenta, Morchella conica, Disciotis venosa, Ophiocordycepssinensis and Cordyceps militaris.

In embodiments, the finished biomat may have a characteristic selectedfrom the group consisting of (a) comprises greater than about 45 wt. %protein content; (b) comprises greater than about 50 wt. % proteincontent; (c) comprises greater than about 55 wt. % protein content; (d)comprises greater than about 60 wt. % protein content; (e) comprisesless than about 5 wt. % RNA content; (f) comprises less than about 4 wt.% RNA content; (g) comprises less than about 3 wt. % RNA content; (h)comprises less than about 2 wt. % RNA content; and (i) combinations ofone of a-d and one of e-h.

In embodiments, the finished biomat may comprise less than about 10 ppmof a mycotoxin selected from the group consisting of Alfatoxin B1,Alfatoxin B2, Alfatoxin G1, Alfatoxin G2, Fumonisin B1, Fumonisin B2,Fumonisin B3, Ochratoxin A, Nivalenol, Deoxynivalenol, Acetyldeoxynivalenol, Fusarenon X, T-2 Toxin, HT-2 Toxin, Neosolaniol,Diacetoxyscirpenol zearalenone, beauvericin, fusarin C, fusaric acid,and any combinations thereof.

In embodiments, the finished biomat may comprise less than about 10 ppmtotal mycotoxin content.

In embodiments, the finished biomat may comprise less than about 5 ppmtotal mycotoxin content.

In embodiments, the finished biomat may comprise greater than about 15wt. % of branched chain amino acids.

It is another aspect of the present invention to provide a biomat of atleast one filamentous fungus, having a dry-mass density of at leastabout 75 grams per liter.

In embodiments, the biomat may comprise greater than about 40 wt %protein and less than about 8 wt % RNA.

In embodiments, the at least one filamentous fungus may belong to anorder selected from the group consisting of Mucorales, Ustilaginales,Russulales, Polyporales, Agaricales, Pezizales and Hypocreales.

In embodiments, the at least one filamentous fungus may belong to afamily selected from the group consisting of Mucoraceae, Ustilaginaceae,Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae,Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae,Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae,Nectriaceae, Bionectriaceae, and Cordycipitaceae.

In embodiments, the at least one filamentous fungus is selected from thegroup consisting of Rhizopus oligosporus, Ustilago esculenta, Hericululmerinaceus, Polyporous squamosus, Grifola frondosa, Hypsizygus marmoreus,Hypsizygus ulmarius (elm oyster), Calocybe gambosa, Pholiota nameko,Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata,Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (pearl),Pleurotus ostreatus var. columbinus (Blue oyster), Tuber borchii,Morchella esculenta, Morchella conica, Morchella importuna, Sparassiscrispa (cauliflower), Fusarium venenatum, strain MK7 (ATCC AccessionDeposit No. PTA-10698), Disciotis venosa, and Cordyceps militaris.Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinulaedodes, Pleurotus djamor, Pleurotus ostreatus, and Leucoagaricus spp.

In embodiments, the biomat may have a characteristic selected from thegroup consisting of (a) comprises greater than about 45 wt. % proteincontent; (b) comprises greater than about 50 wt. % protein content; (c)comprises greater than about 55 wt. % protein content; (d) comprisesgreater than about 60 wt. % protein content; (e) comprises less thanabout 5 wt. % RNA content; (f) comprises less than about 4 wt. % RNAcontent; (g) comprises less than about 3 wt. % RNA content; (h)comprises less than about 2 wt. % RNA content; and (i) combinations ofone of a-d and one of e-h.

In embodiments, the biomat may comprise less than about 10 ppm of amycotoxin selected from the group consisting of Alfatoxin B1, AlfatoxinB2, Alfatoxin G1, Alfatoxin G2, Fumonisin B1, Fumonisin B2, FumonisinB3, Ochratoxin A, Nivalenol, Deoxynivalenol, Acetyl deoxynivalenol,Fusarenon X, T-2 Toxin, HT-2 Toxin, Neosolaniol, Diacetoxyscirpenolzearalenone, fusarin C, fusaric acid, and any combinations thereof.

In embodiments, the biomat may comprise less than about 10 ppm totalmycotoxin content.

In embodiments, the biomat may comprise less than about 5 ppm totalmycotoxin content.

In embodiments, the biomat may comprise greater than about 15 wt. % ofbranched chain amino acids.

In embodiments, the biomat may be produced by the methods describedherein.

It is another aspect of the present invention to provide a method forproducing a biomat of a filamentous fungus, comprising inoculating afilamentous fungus in a bioreactor, wherein the bioreactor comprises acontainer; at least one mesh scaffold disposed within or on a surface ofthe container, the at least one mesh scaffold comprising a first surfaceand a second surface, wherein either or both of the first and secondsurfaces are adapted to receive thereon the inoculum of the filamentousfungus; and a feedstock for the growth of a filamentous fungus,contacting the first surface of the mesh scaffold.

In embodiments, the mesh scaffold may comprise a nylon material.

It is another aspect of the present invention to provide a cultured foodproduct, comprising particles of a filamentous fungus belonging to anorder selected from the group consisting of Mucorales, Ustilaginales,Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, whereinthe filamentous fungus comprises greater than about 40 wt. % proteincontent and less than about 8 wt. % RNA content; and a microbial foodculture.

In embodiments, the microbial food culture may comprise lactic acidbacteria.

It is another aspect of the present invention to provide a method formaking a cultured food product, comprising inoculating particles of afilamentous fungus with a microbial food culture, wherein thefilamentous fungus belongs to an order selected from the groupconsisting of Mucorales, Ustilaginales, Russulales, Polyporales,Agaricales, Pezizales, and Hypocreales, wherein the filamentous funguscomprises greater than about 40 wt. % protein content and less thanabout 8 wt. % RNA content.

In embodiments, the microbial food culture may comprise lactic acidbacteria.

In embodiments, cultured food products described herein may be made bymethods of making cultured food products described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Growth of Fusarium strain MK7 biomat in nutrient medium that wasrefreshed daily after the initial 4-day biomat growth stage.

FIG. 2. Three-centimeter-thick biomat of Fusarium strain MK7 that wasformed in liquid nutrient medium that was refreshed daily (after day 4).Nylon mesh screen underneath the biomat is shown and used for liftingand moving the biomat to fresh medium.

FIG. 3. Continuous flow system designed to continuously feed Fusariumstrain MK7 biomat growth and remove nutrients from media. Biomats shownin channels after 7 days of growth from the time of inoculation.

FIG. 4. Biomat growth after 10 days of growth from the time ofinoculation (6 days under continuous flow +4 days under quiescent/staticconditions).

FIG. 5. Semi-continuous production of biomat showing (A) removal of themost mature portion of the biomat at day 12. After harvesting ⅓ of themost mature biomat at the lower end of the tray, the remaining biomat isphysically moved down in the direction of the arrow until the edge ofthe biomat touches the end of the tray (B). Moving the biomat creates afresh open space at the upper end of the tray where new biomat forms.

FIG. 6. Cumulative production of biomass over time using thesemi-continuous production method. Dashed line is the linear regressionline for day 5 through day 19 (y=0.57x−1.52, r²=0.973). Error bars arestandard deviations of the mean of three replicate trays. Error bars arenot visible when smaller than data point symbol.

FIG. 7. Continuous production of biomat showing removal of the mostmature portion of the biomat at the right. While continuously harvestingthe most mature biomat at the right side of the tray, fresh open spaceis created at the left end of the tray enabling new biomat to form.Liquid medium in the tray can be replenished and/or augmented asrequired or continuously.

FIG. 8. Orange pigmentation of Fusarium strain MK7 biomats (two exciseddisks at the right) after irradiation with UVB light for four hours. Twoexcised disks from non-irradiated control biomats are shown at the left.

FIG. 9. Field emission scanning electron microscopy of 4 day oldFusarium strain MK7 (ATCC Accession Deposit No. PTA-10698) biomatsproduced using MK7-1 medium (described in PCT/US2017/020050) withglycerol, corn starch and corn steep liquor. Images A, B and C showbiomat with extracellular matrix (ECM) removed by ethanol washing. A)View of top surface of biomat with aerial hyphae. B) Cross-section ofthe dense bottom layer with arrow delineating the layer. Thecross-sectional view was created by cutting the biomat with a razorblade. The bottom of the biomat is shown at the bottom left corner ofthe image and the poorly adhering transition layer above the densebottom layer is shown at the upper right corner. C) View of bottomsurface of biomat. D) View of bottom surface of biomat with ECM in place(i.e., ECM not removed with ethanol wash).

FIG. 10. Transmitted light microscope images (100×) of biomats grown onglycerol, starch and corn steep liquor. The image at the left of theaerial hyphal layer reveals the predominant near-vertical orientation ofthe filaments. The image at the right shows the dense bottom layer andthe adjacent transitional layer.

FIG. 11. Biomats produced using the disclosed method. A: Reishimushroom; B: Pearl Oyster mushroom; C: Blue Oyster mushroom; D:Cauliflower mushroom; E: Elm oyster mushroom; F: Giant Puffballmushroom.

FIG. 12. Growth of biomat in the encapsulated reactor starts when cellsattach to the gas-permeable membrane where oxygen is readily available.Over time, biomat grows downward and ultimately fills the space of thereactor, consuming all liquid and nutrients.

FIG. 13. Fusarium strain MK7 biomats grown in five days under staticconditions in Petri dishes covered with semi-permeable membranesconstructed with (A)-(C) polypropylene and (D) polycarbonate.Essentially no free liquid remained in the Petri dish and all nutrientswere incorporated into the biomat. The void/liquid volume of the reactorwas essentially filled with biomat.

FIG. 14. An attached bag separated from the liquid medium by agas-permeable membrane is used to supply and capture gasses. Theintegrated multi-functional membrane allows for ingress of oxygen andegress of CO₂ and other produced gases. Fungal biomass grown in thelower liquid compartment (yellow) converts the feedstocks and nutrientsinto biomat that fills the compartment as it grows. The denseconsolidated biomat can be easily harvested by opening the reactorclosure system (e.g. Zip-lock® type) and removal from the bag.

FIG. 15. Basic reactor (1). Multiple channels (4) with sharedwalls/baffles (9), front valves (6) and back valves (8) and a gaspermeable membrane (2) are shown.

FIG. 16. Basic hermetic reactor (1) with a single gas collection chamber(14).

FIG. 17. Basic hermetic reactor (1) with channeled gas collectionchambers (15, 20).

FIG. 18. Basic hermetic reactor (1) with channeled gas collectionchambers (15) having gas specific channels (30, 40) with gas specificpermeable membranes (2, 50).

FIG. 19. Basic hermetic reactor (1) with cylindrical channels (4),walls/baffles (9), front valves (6) and back valves (8) and a gaspermeable membrane (2).

FIG. 20. Refractive index, density and particle size analysis in10-1000× diluted samples of a vegan milk made with a filamentous fungus.

FIGS. 21A and 21B. Structure of vegan milk under optical microscope at10× magnification and 100× magnification, respectively.

FIG. 22. Plot of viscosity of vegan milk sample against shear rate.

FIG. 23. Generalized schematic of various bioreactor configurations.

FIGS. 24A and 24B. Illustration of hermetic embodiment of bioreactorconfiguration “1” before and after production of a biomat, respectively.

FIG. 25. Illustration of hermetic embodiment of bioreactor configuration“4.”

FIG. 26. Biomat growth process of bag reactor.

FIG. 27. Illustration of hermetic embodiment of bioreactor configuration“4.”

FIGS. 28A and 28B. Illustration of bioreactor utilizing “biomembrane” 3days and 6 days after inoculation, respectively.

FIG. 29. Exemplary schematic of bioreactor including photosynthesizingcyanobacteria.

FIGS. 30A and 30B. Macroscopic and microscopic illustrations,respectively, of biomats produced in feedstock comparison test.

FIGS. 31A and 31B. An illustration of an embodiment of a continuouslyfed, backpressure-eliminating bioreactor and a generalized schematic forcontinuously fed, backpressure-eliminating bioreactors, respectively.

FIG. 32. Illustration of biomat growth in membrane-bag biofilm reactor.

FIG. 33. Illustration of nylon net filter membrane.

FIG. 34. Comparison of biomats grown in light and dark conditions.

FIG. 35A. Illustration of membrane envelope bioreactor (MEBR).

FIG. 35B. Inset of FIG. 35A illustrating a detail of the membraneenvelope bioreactor.

FIG. 36. Illustration of biomat growth rates in the trays with andwithout refreshing growth medium.

DETAILED DESCRIPTION

As used herein, the term “biomat,” unless otherwise specified, refers tocohesive mass of filamentous fungal tissue comprising a network ofinterwoven hyphae filaments. Biomats as that term is used herein may,but need not, be characterized by one or more of a density of betweenabout 50 and about 200 grams per liter, a solids content of betweenabout 5 wt % and about 20 wt %, and sufficient tensile strength to belifted substantially intact from the surface of a growth medium.

As used herein, the term “extracellular matrix,” unless otherwisespecified, refers to extracellular material that at least partiallysurrounds a filamentous fungal structure in a biomat and protects,supports, and/or isolates fungal mycelia of the biomat against asurrounding environment. Extracellular matrices as that term is usedherein may generally include various macromolecules, including but notnecessarily limited to proteoglycans (e.g. heparin sulfate, chondroitinsulfates, keratan sulfates), non-proteoglycan polysaccharides (e.g.hyaluronic acid), and proteins (e.g. collagen, elastin).

Edible filamentous fungi can be used as a nutrition source, such as forprotein, either alone or incorporated into foodstuffs.

While the fruiting bodies of Basidiomycota and Ascomycota filamentousfungi, are used in foodstuffs, there are only a few products primarilycomprising the vegetative mycelia of either the Basidiomycota orAscomycota filamentous fungi. This is due, in part, to mycelia typicallybeing either subterraneous or largely inseparable from the matter onwhich it grows.

Yet under particular conditions, filamentous fungi can form fungalbiomats via surface fermentation under anaerobic, microaerobic, oraerobic conditions or a combination thereof. Here, the filamentousfungal biomats comprise the fungal species and/or strain and/or progenythereof primarily in the form of mycelia, fragments of mycelia, hyphae,fragments of hyphae, and to a lesser extent contain conidia,microconidia, macroconidia, or any and all combinations thereof and insome cases can also contain pycnidia, chlamydospores, and portions ofextracellular matrix.

Typically, the filamentous fungal biomats are primarily comprised ofmycelia; that is, a complex network of interwoven vegetative hyphaefilaments. The average length of non-broken filaments within the biomatis generally at least 0.1 mm, such as between 0.1 mm-100 cm, or anyrange defined by any two whole numbers between 1 mm and 100 cm. In someembodiments, the average length can be at least 0.1 mm, 0.25 mm, 0.5 mm,1.0 mm, 1.4 mm 1.6 mm, 1.7 mm, 1.8 mm, 2 mm, 2.5 mm, 5 mm, 2 cm, 3 cm, 4cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85cm, 90 cm, 85 cm, or 100 cm, or any number in between.

Described herein are food materials comprising particles of ediblefilamentous fungi and particularly ones that are grown as biomats beforebeing processed into particles.

The filamentous fungi suitable for use in the invention (either asbiomats or as particles in food materials) may be selected from thephyla or divisions zygomycota, glomermycota, chytridiomycota,basidiomycota or ascomycotal. The phylum (or division) basidiomycotacomprises, inter alia, the orders Agaricales, Russulales, Polyporalesand Ustilaginales; the phylum ascomycota comprises, inter alia, theorders Pezizales and Hypocreales; and the phylum zygomycota comprises,inter alia, the order Mucorales. The particles of edible filamentousfungi of the present invention belong to an order selected fromUstilaginales, Russulales, Polyporales, Agaricales, Pezizales,Hypocreales and Mucorales.

In some embodiments, the filamentous fungi of the order Ustilaginalesare selected from the family Ustilaginaceae. In some embodiments, thefilamentous fungi of the order Russulales are selected from the familyHericiaceae. In some embodiments, the filamentous fungi of the orderPolyporales are selected from the families Polyporaceae or Grifolaceae.In some embodiments, the filamentous fungi of the order Agaricales areselected from the families Lyophyllaceae, Strophariaceae, Lycoperdaceae,Agaricaceae, Pleurotaceae, Physalacriaceae, or Omphalotaceae. In someembodiments, the filamentous fungi of the order Pezizales are selectedfrom the families Tuberaceae or Morchellaceae. In some embodiments, thefilamentous fungi of the order Mucorales are selected from the familyMucoraceae.

In some embodiments, the filamentous fungi may be selected from thegenera Fusarium, Aspergillus, Trichoderma, and Rhizopus.

Examples of the species of filamentous fungi include, withoutlimitation, Ustilago esculenta, Hericululm erinaceus, Polyporoussquamosus, Grifola fondrosa, Hypsizygus marmoreus, Hypsizygus ulmariuos(elm oyster) Calocybe gambosa, Pholiota nameko, Calvatia gigantea,Agaricus bisporus, Stropharia rugosoannulata, Hypholoma lateritium,Pleurotus eryngii, Pleurotus ostreatus (pearl), Pleurotus ostreatus var.columbinus (Blue oyster), Tuber borchii, Morchella esculenta, Morchellaconica, Morchella importuna, Sparassis crispa (cauliflower), Fusariumvenenatum, strain MK7 (ATCC Accession Deposit No. PTA-10698), Disciotisvenosa, Cordyceps militaris, Ganoderma lucidum (reishi), Flammulinavelutipes, Lentinula edodes, Ophiocordyceps sinensis. Additionalexamples include, without limitation, Trametes versicolor, Ceriporialacerate, Pholiota gigantea, Leucoagaricus holosericeus, Pleurotusdjamor, Calvatia fragilis, Handkea utriformis, and Rhizopus oligosporus.

In some embodiments, the filamentous fungus is a Fusarium species. Insome embodiments, the filamentous fungus is the Fusarium strain MK7(ATCC PTA-10698 deposited with the American Type Culture Collection,1081 University Boulevard, Manassas, Va., USA). Strain MK7 waspreviously reported to be a Fusarium oxysporum strain. However, it hassubsequently been identified as not being a oxysporum strain. In someembodiments, the filamentous fungus is the Fusarium strain Fusariumvenenatum.

As described in detail herein, the filamentous fungi of the presentinvention have a surprisingly high protein content. It is noted that thefilamentous fungi that grow naturally or in the wild or by prior artmethods do not possess such high protein contents, whereas filamentousfungi grown or cultured as disclosed herein have a high protein content,and in particular higher protein content than is achieved in nature or,for some fungi by prior fermentation methods. For example, proteincontents of filamentous fungi described herein refer to the proteincontents of the filamentous fungi as grown in a biomat according to thepresent disclosure. Consequently, food materials of the invention havehigh protein contents based on the filamentous fungi components of thematerials without the need for and/or in the absence of protein contentfrom a non-filamentous fungal source. Thus, in various embodiments, foodmaterials of the invention do not contain or have an absence of proteincontent from a non-filamentous fungal source.

In some embodiments, the filamentous fungi comprise at least about 30wt. % protein content. Unless specified otherwise herein, percentages ofcomponents, such as proteins, RNA or lipids, of biomats or filamentousfungi particles, are given as a dry weight percent basis. For example,biomats can be dried for 2 days at 99° C. and further air dried for afew days, at the end of which the biomats are expected to contain about5 wt. % or less moisture, such as less than 4 wt. %, less than 3 wt. %,less than 2 wt. %, less than 1 wt. %, less than 0.5 wt. %, less than 0.1wt. % moisture. The total protein content in dried biomat samples can bemeasured using total nitrogen analysis method for estimating proteins.

In some embodiments, the filamentous fungi comprise at least about 30%,at least about 31 wt. %, at least about 32 wt. %, at least about 33 wt.%, at least about 34 wt. %, at least about 35 wt. %, at least about 36wt. %, at least about 37 wt. %, at least about 38 wt. %, at least about39 wt. %, at least about 40 wt. %, at least about 41 wt. %, at leastabout 42 wt. %, at least about 43 wt. %, at least about 44 wt. %, atleast about 45 wt. %, at least about 46 wt. %, at least about 47 wt. %,at least about 48 wt. %, at least about 49 wt. %, at least about 50 wt.%, at least about 51 wt. %, at least about 52 wt. %, at least about 53wt. %, at least about 54 wt. %, at least about 55 wt. %, at least about56 wt. %, at least about 57 wt. %, at least about 58 wt. %, at leastabout 59 wt. %, at least about 60 wt. % protein content, at least about61 wt. %, at least about 62 wt. %, at least about 63 wt. %, at leastabout 64 wt. %, at least about 65 wt. %, at least about 66 wt. %, atleast about 67 wt. %, at least about 68 wt. %, at least about 69 wt. %,at least about 70 wt. % protein content, at least about 71 wt. %, atleast about 72 wt. %, at least about 73 wt. %, at least about 74 wt. %,at least about 77 wt. %, at least about 76 wt. %, at least about 77 wt.%, at least about 78 wt. %, at least about 79 wt. %, or at least about80 wt. % protein content. Alternatively, in embodiments of theinvention, filamentous fungi can comprise protein in a range between 30wt. % and 80 wt. or in any whole number percentage range between 30 wt.% and 80 wt. %. See Examples 21-23.

The filamentous fungi of the present invention also have surprisinglylow RNA content. High amounts of RNA in food have been shown to haveadverse health or physiological effects. For example, diets that arehigh in purines (present in RNA) are associated with incidence of gout.Filamentous fungi as grown or cultured as disclosed herein haveintrinsically low RNA content and do not require additional orsupplemental treatment to modify or lower the RNA content. Thus, invarious embodiments, food materials of the invention do not containfilamentous fungal components that have significant levels of RNA and/orthat have been treated for the purpose of modifying or lowering the RNAcontent of the components or food materials. In other embodiments, itwill be recognized that food materials of the invention havingfilamentous fungal components with naturally low amounts of RNA may betreated, for example by heating or steaming to inactive the fungus, orby other treatments that would also reduce or degrade RNA, if it werepresent. Such materials can be characterized as having filamentousfungal components that have a low RNA content as described herein priorto or without any such reduction or degradation of RNA.

In some embodiments, the filamentous fungi comprise less than about 8wt. % RNA content. The wt. % RNA content is given on a dry weight basis.For example, the total RNA content in dried biomat samples can bemeasured using the purine analysis method. See Example 24.

In some embodiments, the RNA content in the filamentous fungi is lessthan about 8.0 wt. % RNA content, less than about 7 wt. % RNA content,less than about 6 wt. % RNA content, less than about 5.0 wt. % RNAcontent, less than about 4 wt. % RNA content, less than about 3 wt. %RNA content, less than about 2 wt. % RNA content, or less than about 1wt. % RNA content, or alternatively less than any increment 0.1-wt %increment less than about 8.0 wt %. Alternatively, in embodiments of theinvention, filamentous fungi can comprise RNA in a range between 0.5 wt.% and 8 wt. % or any sub-range thereof.

In some embodiments, the filamentous fungus comprises a high proteincontent combined with a low RNA content as described above. For example,in some embodiments the filamentous fungus may comprise greater than 45wt. % protein, greater than 50 wt. % protein, greater than 55 wt. %protein, or greater than 60 wt. % protein and less than about 8 wt. %RNA content, less than about 5 wt. % RNA content, less than about 4 wt.% RNA content, less than about 3 wt. % RNA content, or less than about 2wt. % RNA content. In other embodiments, the filamentous fungus can haveany protein content described above in combination with any RNA contentdescribed above.

The filamentous fungi of the present invention and related foodmaterials can also be characterized as having surprisingly low mycotoxincontent. Known mycotoxins include Alfatoxin B1, Alfatoxin B2, AlfatoxinG1, Alfatoxin G2, Fumonisin B1, Fumonisin B2, Fumonisin B3, OchratoxinA, Nivalenol, Deoxynivalenol, Acetyl deoxynivalenol, Fusarenon X, T-2Toxin, HT-2 Toxin, Neosolaniol, Diacetoxyscirpenol, beauvericin, fusarinC, fusaric acid, and zearalenone. In some embodiments, the total amountof mycotoxins and/or the total amount of any one of or subset of theabove-listed mycotoxins in a filamentous fungi, biomat or food materialof the invention is less than about 10 ppm. In other embodiments, thetotal amount of mycotoxins and/or the total amount of any one of orsubset of the above-listed mycotoxins is less than about 9 ppm, lessthan about 8 ppm, less than about 7 ppm, less than about 6 ppm, lessthan about 5 ppm, less than about 4 ppm, less than about 3 ppm, lessthan about 2 ppm, less than about 1 ppm, less than about 0.9 ppm, lessthan about 0.8 ppm, less than about 0.7 ppm, or less than about 0.6 ppm.See Example 25.

The filamentous fungi of the present invention also have a surprisinglyhigh branched amino acid content. Branched amino acids refer to leucine,isoleucine and valine. In some embodiments, the total amount of branchedamino acids is greater than about 10 wt. %, greater than about 11 wt. %,greater than about 12 wt. %, greater than about 13 wt. %, greater thanabout 14 wt. %, greater than about 15 wt. %, greater than about 16 wt.%, greater than about 17 wt. %, greater than about 18 wt. %, greaterthan about 19 wt. %, greater than about 20 wt. %, greater than about 21wt. %, greater than about 22 wt. %, greater than about 23 wt. %, greaterthan about 24 wt. %, greater than about 25 wt. %, greater than about 26wt. %, greater than about 27 wt. %, greater than about 28 wt. %, greaterthan about 29 wt. %, greater than about 30 wt. %. See Example 23 for thecontent of branched amino acids in exemplary strain MK7 and Fusariumvenenatum biomats. Example 23 also shows the fatty acid profile of thetwo fungi.

Growing and Harvesting Filamentous Fungal Biomats

The growth of filamentous fungal biomats can be accomplished via surfacefermentation. This involves inoculating a liquid medium containing acarbon source and a nitrogen source with filamentous fungal cells.Suitable carbon sources are sugars (e.g. sucrose, maltose, glucose,fructose, Japan rare sugars, etc.), sugar alcohols (e.g. glycerol,polyol, etc.), starch (e.g. corn starch, etc.), starch derivative (e.g.maltodextrin, cyclodextrin, glucose syrup, hydrolysates and modifiedstarch), starch hydrolysates, hydrogenated starch hydrolysates (HSH;e.g. hydrogenated glucose syrups, maltitol syrups, sorbitol syrups,etc.), lignocellulosic pulp or feedstock (e.g. sugar beet pulp,agricultural pulp, lumber pulp, distiller dry grains, brewery waste,etc.), corn steep liquors, acid whey, sweet whey, milk serum, wheatsteep liquors, carbohydrates, food waste, olive oil processing waste,hydrolysate from lignocellulosic materials, and/or combinations thereof.The filamentous fungi generate biomats which are located on the surfaceof the growth media.

Liquid growth media according to the present invention may becharacterized by a desired or preselected mass ratio of carbon tonitrogen (“C:N ratio”). Typically, the C:N ratio of liquid growth mediaaccording to the present invention may have a C:N ratio of between about1:1 and about 50:1, or between about 2.5:1 and about 30:1, or betweenabout 5:1 and about 10:1, or between about any ratio between 1:1 and50:1 and about any other ratio between 1:1 and 50:1. By way ofnon-limiting example, growth media according to the present inventionmay have a C:N ratio of about 2.5:1, about 5:1, about 7.5:1, about 10:1,about 12.5:1, about 15:1, about 17.5:1, about 20:1, about 22.5:1, about25:1, about 27.5:1, about 30:1, about 32.5:1, about 35:1, about 37.5:1,about 40:1, about 42.5:1, about 45:1, about 47.5:1, or about 50:1, oralternatively about any ratio of the form X:2 where X is an integerbetween about 2 and about 100.

Inoculation may be done with an inoculum comprising planktonicfilamentous fungal cells, conidia, microconidia or macroconidia orspores, or fruiting bodies. In many cases, especially for Ascomycotafungi, growth media may be inoculated with an inoculum comprisingplanktonic filamentous fungal cells, conidia, microconidia ormacroconidia. Ideally, the cells of the inoculum float on the surface ofthe growth media, such as those cells having a high lipid content, andresult in increased growth rate. Cells or clumps of cells that aresubmersed within the growth media can negatively affect the cellsfloating on the surface and the biomats they form. Specifically, thebiomats resulting from growth media containing a significant number ofclumped submerged cells are typically discolored and tend to not growhomogeneously dense mats.

In some embodiments, the inoculum may comprise spores. For example, inone embodiment, approximately 2 cc of sterile Basidiomycota sporessuspended in deionized water from a spore syringe (e.g. MycoDirect,Huntley, Ill.) were used to inoculate approximately 75 mL of growthmedia in small Pyrex trays. Alternatively, 1 cc of spores suspended indeionized water from a spore syringe was plated on a container havingmalt extract agar media+CF (30 g dry malt extract, 20 g agar, 1000 mLwater+0.01% chloramphenicol) using standard sterile conditions.Containers were sealed with parafilm and incubated at room temperatureuntil mycelium completely covered the surface of the agar. A segment ofmycelium from the agar preparation approximately 2 cm in width cut intoa wedge was then diced into the smallest size possible beforetransferring to a tube with growth media. Liquid culture tubes weresealed, incubated at room temperature, and shaken by hand or shaken bymechanical means (i.e. continuous shaking or a continuous stirred tankreactor) for about 1 minute at least five (5) times per day to break upmycelium as much as possible. Liquid cultures were incubated untilvisually turbid, typically three or more days. The liquid cultures werethen used to inoculate growth medium in trays at a 10% or 15% of totalgrowth medium volume.

In some embodiments, the inoculum may comprise fruiting bodies. Forexample, in some embodiments, Basidiomycota fruiting bodies were used togenerate inoculum for initiating filamentous biomats. In some instances,inoculum was prepared by (a) surface sterilizing fruiting bodies, forexample in a 5% bleach solution, (b) rinsing with sterile media, (c)grinding under sterile conditions to either less than 5 mm longaggregates or greater than 5 mm aggregates, depending on the final use,(d) surface sterilizing the ground mushroom biomass for example in a 5%bleach solution, and again rinsing with sterile media. 5 grams of theground surface-sterilized fruiting body biomass was used directly asinoculum. In other instances, a pure culture derived from a fruitingbody was used. In this instance, ˜3 mm³ portions of fruiting body wasplaced on agar media containing 0.01% chloramphenicol and incubated atroom temperature. After 2-5 days of growth, hyphae were transferred ontofresh agar +chloramphenicol media and grown for another 3-7 days.Culture purity was confirmed by extracting and purifying DNA (FastDNASpin Kit, MP Biomedicals), sequencing the 18S rRNA sequence and/or ITSregion, and performing phylogenetic classification of the sequencesusing Blast (NCBI database). Upon confirmation, hyphae were used toinoculate 50 mL of sterile liquid media and agitated/rotated at 185 rpmfor approximately 5 days before using as inoculum at a ratio of about7.5% inoculum to 92.5% liquid media.

While a number of different media can be used, certain media performbetter than others for growth of filamentous fungal biomats; by way ofnon-limiting example, Hansen's media (per liter=1.0 g peptone, 0.3 gKH₂PO₄.7H₂O, 2.0 g MgSO₄.7H₂O 5.0 g glucose with a C:N ratio of 26.9)did not yield full, cohesive biomats, while those media which workexceptionally well include MK7A, MK7-1, MK7-3 (all described in WO2017/151684), as well as the media presented below. These are alsodescribed in Example 12.

Malt Medium 001 (C:N ratio of 19.1) Ingredient Amount Grade LightPilsner Malt 40.0 g Food Peptone 4.0 g Research Yeast Extract Powder 1.2g Research Canola Oil 1.0 mL Food Ground Oats 4.0 g Food Tap H₂O 1000 mLN/A

MK-7 SF Medium (C:N ratio of 7.5) Ingredient Amount Grade NH₄NO₃ 7.553 gACS Urea 2.548 g USP CaCl₂ 2.000 g Reagent MgSO₄ * 7H₂O 2.000 g USPKH₂PO₄ 7.500 g Reagent Trace * 2.000 mL * Glycerol 0.075 Kg Food/USPYeast Extract 1.750 g Research FeCL₂ * 4H₂O 0.020 g Reagent DI H₂O 0.940L N/A

Trace Components * Micronutrients* mg/L Grade FeSO4•7 H2O 9.98 ACSZnSO4•7 H2O 4.4 USP/FCC MnCl2•4 H2O 1.01 Reagent CoCl2•6 H2O 0.32Reagent CuSO4•5 H2O 0.31 Technical (NH4)6Mo7O24•4 H2O 0.22 ACS H3BO30.23 ACS EDTA, free acid 78.52 Electrophoresis

Malt Media 001 Supplemented with NH₄NO₃ (C:N ratio of 7.5) IngredientAmount Grade NH₄NO₃ 5.0 g ACS Light Pilsner Malt 40.0 g Food Peptone 4.0g Research Yeast Extract Powder 1.2 g Research Canola Oil 1.0 mL FoodGround Oats 4.0 g Food Tap H₂O 1000 mL N/A

Osmotic concentrations as osmolality can be determined by measurement ofmedia with an Osmometer (e.g., Model 3250 SN: 17060594) capable ofmeasuring up to 5000 mOsm/kg. Three readings were taken for severalmedia and provided the following results: Hansen's=39, 39, 38; Malt001=169, 168, 169; MK-7 SF=1389, 1386, 1387; Malt 001+NH₄NO₃=288, 287,286.

As noted before, methods of the invention can result in increasing theprotein content, amino acid profile (e.g. content of branched-chainamino acids), and/or nutritional content of the filamentous fungus.Without being bound by theory, this result is believed to be due in partto the media used to grow the fungus.

For example, while the natural protein content of the fruiting body ofBlue Oyster mushrooms (Pleurotus ostreatus var. Columbinus) is reportedto be about 16.32% (Ulziijargal and Mau (2011) Int J MedicinalMushrooms, 13(4):343-49) or 24.65% (Stamets (2005) Int J MedicinalMushrooms 7:103-110), as shown in Example 12, Blue Oyster biomats grownaccording to the present invention on Malt 001 media have a highermoisture corrected protein content of 29.82%, an increase in proteincontent of 13.6% or 5.71%.

The protein content of the fruiting body of Pearl Oyster mushrooms(Pleurotus ostreatus) is reported to be about 23.85% (Ulziijargal andMau (2011) Int J Medicinal Mushrooms, 13(4):343-49) or 27.25% (Stamets(2005) Int J Medicinal Mushrooms 7:103-110); Pearl Oyster biomats grownaccording to the present invention have a higher moisture correctedprotein content of 39.77%, an increase in protein content of at least46% to a maximum of 67%.

The protein content of the fruiting body of Cauliflower mushrooms(Sparassis crispa) is reported to be about 13.4% (Kimura (2013) BioMedResearch International); Cauliflower biomats grown according to thepresent invention have a higher moisture corrected protein content of32.21%-46.24%, an increase in protein content of least 140% to a maximumof 245%.

Other characteristics of the media that are believed to be important forthe growth of biomats on the surface of a fermentation media are theosmotic pressure and the ionic strength of the media. In someembodiments, the osmotic pressure of the media for growth of biomats canbe greater than about 3 atm, greater than about 10 atm, greater thanabout 20 atm, greater than about 30 atm, greater than about 40 atm,greater than about 50 atm, greater than about 60 atm, greater than about70 atm, greater than about 80 atm, greater than about 90 atm, greaterthan about 100 atm, greater than about 110 atm, greater than about 120atm, greater than about 125 atm, or greater than any whole-numberatmosphere value greater than 3 atmospheres through greater than 125atmospheres. In alternative embodiments, the osmotic pressure may rangebetween about 3 atm to about 125 atm, between about 20 atm and about 100atm or between any two whole number atm values between 3 and 125.

In some embodiments, the ionic strength of the media that can be used togrow biomats can be greater than about 0.02 M, greater than about 0.05M, greater than about 0.10 M, greater than about 0.20 M, greater thanabout 0.30 M, greater than about 0.40 M, greater than about 0.50 M,greater than about 0.60 M, greater than about 0.70 M, greater than about0.80 M, greater than about 0.90 M, greater than about 1.0 M, or greaterthan any one-hundredth M value greater than 0.02 M through greater than1.00 M. In alternative embodiments, the ionic strength may range betweenabout 0.02 M to about 1.0 M, between about 0.10 M and about 0.50 M orbetween any two number molar concentration values between 0.01 and 1.0.

Harvesting of biomats can occur at any time a sufficiently thick biomathas formed. Harvesting typically occurs after 2-3 days of growth,although in some instances longer growth periods are desirable, such aswhen thicker or denser biomats are desired/required. For example,harvesting can occur after growth of between 2 days and 60 days or anyrange of days or partial days (e.g., hours) between 2 days and 60 days.For example, such growth periods can be 3.5-4 days, 3-5 days, 4-6 days,5-7 days, 6-9 days, 7-10 days, or 19-21 days, or alternatively about anywhole number of days up to and including about 21 days. As used herein,the term “harvesting,” refers to any process or step that stops growthof a biomat (e.g., separation from a nutrient source or change intemperature conditions) and/or that modifies a physical characteristicof a biomat (e.g., converting a biomat into particles or strips).

Due to the cohesive structure of the filamentous biomats grown undersurface fermentation conditions described in PCT/US2017/020050 andherein, the filamentous biomats have enough tensile strength to belifted essentially intact from the surface of the media at the end ofthe growth period. In various embodiments, biomats of the invention canhave a tensile strength of at least about 30 g/cm², at least about 40g/cm², at least about 50 g/cm², at least about 60 g/cm², at least about70 g/cm², at least about 80 g/cm², at least about 90 g/cm², at leastabout 100 g/cm², at least about 150 g/cm², at least about 200 g/cm², atleast about 250 g/cm², at least about 300 g/cm², at least about 350g/cm², at least about 400 g/cm², at least about 450 g/cm², at leastabout 500 g/cm², at least about 550 g/cm², or at least about 600 g/cm²,or at least about 650 g/cm², or at least about 700 g/cm², or at leastabout 750 g/cm², or at least about 800 g/cm², or at least about 850g/cm², or at least about 900 g/cm², or at least about 950 g/cm², or atleast about 1000 g/cm², or at least about 1500 g/cm², or at least about2000 g/cm², or at least about 2500 g/cm², or at least about 3000 g/cm²,or at least about 3500 g/cm², or at least about 4000 g/cm². In otherembodiments, biomats of the invention can have a tensile strength ofgreater than any whole number greater than 30 g/cm². Alternatively, thetensile strength of biomats of the invention can be in a range ofbetween about 30 g/cm² and about 4000 g/cm² or any whole number rangebetween about 30 g/cm² and about 4000 g/cm². A suitable method formeasuring tensile strength is explained in Example 41.

Table 1A presents some examples of tensile strength and other physicalcharacteristics measured for various filamentous fungi.

TABLE 1A Average Tensile Strength for some filamentous fungal biomatsAvg. Avg. Tensile Carbon Thickness Width Break wt Strength Organismsource (cm) (cm) (g) (g/cm²) Giant Malt  0.13 1.2 47.12 314.13 PuffballGlycerol 0.10-1.3  1.2 29.05 214.85 MK7-1SF 0.25-0.35 0.65-0.8  30.67263.98 Malt + 0.09-0.10 0.9-1.1 27 281.15 NH₄NO₃ Cauliflower Malt0.15-2.0  1.0-1.2 101.05 507.38 Glycerol 0.09-0.20 1.2 202.17 242.91Reishi Malt 0.5 1.0-1.2 101.05 1854.54 Blue Oyster Malt 0.5 1.2 43.4072.74 Glycerol 0.4 1.3 19.04 37.27 Pearl Oyster Malt 0.5 1.0-1.2 56.798.96 Elm Oyster Malt  0.35 1.2 50.28 143.67 F. strain MK7 Glycerol0.5-0.8 1.0 >742 >570

Table 1B shows additional examples of tensile strength and otherphysical characteristics measured for various filamentous fungi obtainedusing other media.

Ten- Den- sile Wet Dry sity Stren- initial Final Weight Weight g/ Yieldgth pH pH g g cm³ g/m² g/cm² C:N 5 MK-7 3.3 6.2 0.4888 0.2953 0.48483.33 333.33 F. 4.5 6.15 0.3379 0.275 0.14 71.43 186.67 Venen- atum GPB6 6.25 0.527 0.2708 0.21 144.76 562.96 C:N 7.5 MK-7 3.3 7.2 0.827 0.44160.31 314.62 1454.55 F. 4.5 4.81 0.57 0.3245 0.09 89.06 166.67 Venen-atum GPB 6 5.45 0.348 0.2851 0.08 38.41 1259.26 C:N 15 MK-7 3.3 4.910.4833 0.257 0.07 92.31 191.11 F. 4.5 3.49 0.3458 0.2734 0.16 63.81800.00 Venen- atum GPB 6 2.74 0.3245 0.322 0.20 197.12 1559.23 C:N 30MK-7 3.3 2.36 0.2832 0.2774 0.10 103.64 426.67 F. 4.5 3.29 0.323 0.31420.06 76.03 370.37 Venen- atum GPB 6 2.87 0.271 0.2688 0.17 134.91 833.33C:N 40 MK-7 3.3 2.81 1.3952 0.3638 0.05 156.09 312.12 F. 4.5 2.97 0.50970.3637 0.43 215.95 3151.52 Venen- atum GPB 6 3.1 0.7196 0.3487 0.36179.66 1040.00

In various embodiments, biomats of the invention can have a thicknessranging from about 0.05 cm to at least about 2 cm.

In various embodiments, biomats of the invention can have a widthranging from about 0.6 cm to about 3 meters. Generally, biomats producedaccording to the present invention may have a width approximately equalto a width of the vessel in which the biomat is grown.

Surface fermentation can be carried out under various conditions,including static media conditions (as described in PCT Publication WO2017/151684, which is incorporated herein by reference in its entirety),semi-static media conditions, and continuous media flow conditions. Someembodiments are described in Examples 1-4.

Growth under semi-static media conditions means that at least a portionof the medium is replaced before the filamentous fungal biomat isharvested. These conditions allow linear dry biomass production over anextended period of time demonstrating the suitability of this system tooperate as a continuous production system. For example, in oneexperiment, linear dry biomass production was achieved from day 4through day 18 (r²=0.995), after which biomass weight stabilized atabout 2.5 Kg dry/m².

Biomats can also be produced under continuous media flow conditionswhere biomat growth is confined to the surface of the growth media wherethe medium underneath the mat is continuously refreshed orsemi-continuously refreshed.

In some instances, however, it is desirable to harvest the growingbiomat on a semi-continuous basis. Here, removal of some portion of thebiomat occurs and the remaining portion is then physically moved to theopen area of medium that was created by removal of the portion ofbiomat. This can be accomplished by physically grasping the biomat andpulling it until it touches the end of the surface fermentationcontainer or by other mechanical means. The resulting open area is thenavailable for new biomat growth without a separate or additionalinoculation step since the medium already contains viable fungal cells.This process can be repeated periodically, which can be particularlyuseful when the medium is refreshed or nutrients that have becomelimited are reintroduced.

Biomat harvesting can also be done on a continuous basis. Continuousremoval can be facilitated by a number of mechanisms. One such exampleis a roller wheel that is attached to the mature end of the biomat (seeFIG. 7). The roller wheel slowly turns and harvests the mature biomatand at the same time creates open medium for growth of new biomat at theother end of the surface fermentation container. In various embodiments,harvesting can be conducted at a rate of at least about 0.1 cm/day, 0.2cm/day, 0.3 cm/day, 0.4 cm/day, 0.5 cm/day, 0.6 cm/day, 0.7 cm/day, 0.8cm/day, 0.9 cm/day, 1.0 cm/day, 1.1 cm/day, 1.2 cm/day, 1.3 cm/day, 1.4cm/day, 1.5 cm/day, 1.6 cm/day, 1.7 cm/day, 1.8 cm/day, 1.9 cm/day, 2.0cm/day, 2.1 cm/day, 2.2 cm/day, 2.3 cm/day, 2.4 cm/day or 2.5 cm/day. Atypical rate of harvesting is 1.56 cm/day, although this can be alteredfor particular needs or as desired by a user.

In some cases, UVB light (290-320 nm) can trigger pigment production byfilamentous fungi, such as for Fusarium strain MK7 (ATCC AccessionDeposit No. PTA-10698), producing a pigmented biomat. In addition to acolor change, which can be useful for creating various food effects,treatment with UVB converts ergosterol present in the fungal cellmembranes into vitamin D2 and increases production of carotenoids, suchas beta carotene and astaxanthin. Consequently, irradiating filamentousfungi, such as Fusarium strain MK7, with UVB can be used to increasevitamin D2 and carotenoids in the resulting biomats.

In some cases, the filamentous fungal biomats formed are composed oflayers which are uniform in appearance, one surface of the filamentousbiomat in contact with the air and one surface in contact with thesynthetic media. In other cases, at least two distinct layers arepresent: an aerial hyphae layer at the top surface and a densemulticellular bottom layer in contact with the synthetic media.Oftentimes three distinct layers are present: (a) an aerial hyphae layerat the top surface, (b) a dense bottom layer and (c) a transitionallayer between the top and bottom layers. The transitional layer may beonly loosely attached to the dense bottom layer, in those cases enablingeasy separation of the bottom layer from the rest of the biomat.Filament densities of the transitional layer range from slightly lessdense than the bottom layer in the zone where the two layers meet, to adensity that is comparable to the aerial hyphae near the top of thebiomat.

In some embodiments, biomats may comprise strain MK7 (ATCC AccessionDeposit No. PTA-10698), Fusarium venenatum, Rhizopus oligosporus,Morchella esculenta (Morel), Morchella conica (Morel), Morchellaimportuna (Morel), Calvatia gigantea (giant puffball), Pleurotusostreatus (pearl oyster), Pleurotus ostreatus var. columbinus (blueoyster), Sparassis crispa (cauliflower), Ganoderma lucidum (Reishi),Hypsizygus ulmarius (elm oyster).

Inactivation of Filamentous Fungal Biomats

While biomats can be rinsed to remove excess growth media, biomatrinsing is not required, although in some cases the removal of growthmedia or excess growth media is preferable. Similarly, biomats can besqueezed to remove excess growth media, again not required, but whichmay be preferable for some applications.

Elimination of cell viability and the potential of further biomat growthis desired in some instances, such as for use of the biomat as astand-alone protein source or a protein ingredient in foodstuffs. Thiscan be accomplished by heating, irradiation, ethanol and/or steaming.

For the heating process, filamentous fungal biomats can be treatedaccording to WO 95/23843 or British Patent No 1,440,642, for example, orincubated at temperatures that destroy the vast majority of theorganism's RNA without adversely affecting the organism's proteincomposition.

In irradiation, filamentous fungal biomats are exposed to ionizingenergy, such as that produced by ⁶⁰Co (or infrequently by ¹³⁷Cs)radioisotopes, X-rays generated by machines operated below a nominalenergy of 5 MeV, and accelerated electrons generated by machinesoperated below a nominal energy of 10 MeV.

Steaming can also be used for inactivating some filamentous fungalbiomats, such as those produced by Fusarium strain MK7 (ATCC AccessionDeposit No. PTA-10698) and F. venenatum, as steaming can also removesome specific metabolites from the biomat construct if those metabolitesare produced. Here, biomats are placed such that biomat excreted liquidsand condensed steam can easily drip away from the biomats. Suitablebiomat holding systems include porous plastic mesh and porous trays.Other biomat holding systems include, but are not limited to, systemsthat secure the biomat in a vertical position, such as systems with aclamping mechanism that clamps at least one end of a biomat while theremaining end(s) of the biomat hang from said clamp and mesh systemswhich clamp at least two sides of the biomat, to name but a few.

Biomats are positioned within a steamer such that heated steam, such assteam of a temperature greater than 85° C. or 95° C., comes into contactwith the biomats. In those cases where multiple trays are placed in asingle steamer, for example one tray above the other, it is preferred toprotect a lower positioned biomat from the drippings of a higherpositioned biomat. Protection should be of a form which allows steam tocontact biomats, thereby de-activating biomat viability, and to alsodeflect biomat excreted liquids and condensed steam produced at a higherlevel in the steamer from contacting biomats positioned at a lower levelin the steamer. In one embodiment, a cone is positioned between an uppertray and a lower tray to accomplish this result. In other embodiments,separation between upper and lower trays also include at least one othergeometric shape such as a cylinder, a cube and/or cuboid, a pyramid, asphere, a tori, and/or other platonic solids. In yet another embodiment,trays are separated using at least one cylinder, cube and/or cuboid,pyramid, sphere, tori, other platonic solid, mesh, porous belt, orcombinations thereof.

Biomats are steamed at least to the point where biomat viability isreduced such that further biomat growth and/or cellular reproductionwithin a biomat is negligible. Biomat viability is a function of theoriginal substrate, biomat development, steam/heat transfercharacteristics, biomat position in a steamer and biomat orientationrelative to evolved steam. As an example, Fusarium strain MK7 biomatsgrown on a glycerol or acid whey substrate are non-viable after 5minutes, and in some cases less than 5 minutes, of steaming. Steamedmats can be rinsed and/or squeezed to remove mat excretions andcondensed steam.

The inactivated edible filamentous fungal biomats can be used directlyas a protein source, for example in preparing foodstuffs largelycomparable to tofu, bacon, and jerky, to name but a few.

Particles of Filamentous Fungal Biomats

The inactivated edible filamentous fungal biomats can also be sizereduced for use as a protein source in foodstuffs. The size reductioncan occur by mechanical means such as cutting, chopping, dicing,mincing, grinding, blending, etc. or via sonication and is conductedprior to mixing with other ingredients or liquids. Size reducedparticles can be uniform in size or variable.

Typically, the length of the sized reduced particles is between 0.05-500mm, the width is between 0.03-7 mm, and height is between 0.03-1.0 mm.For example, flour-type particles typically range between 0.03 mm and0.4 mm, jerky-type particles range between 100 mm and 500, etc. Largersize particles can be produced. For example, biomats have been grown ininflatable pools (66″ in diameter) producing a single biomat 66″ indiameter and completely round. Larger vessels can be used to grow evenlarger mats.

The number of size reduced particles produced per biomat is dependent onthe initial biomat size and the purpose for which the biomat sizereduced particles will be used.

Large Particles

In some embodiments, the inactivated edible filamentous fungal biomatsare reduced to particles, wherein at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90% of the particles have aparticle length of about 0.05 mm to about 500 mm, a particle width ofabout 0.03 mm to about 7 mm, and a particle height of about 0.03 mm toabout 1.0 mm, or alternatively in any subranges within these ranges. Forexample, at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90% of the particles may have a particle length ofabout 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm toabout 200 mm; a particle width of about 0.05 mm to about 2 mm, or about1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle heightof about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, orabout 0.08 mm to about 1.0 mm.

In some embodiments, the inactivated edible filamentous fungal biomatsare reduced to particles, wherein at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90% of the mass of the particleshave a particle length of about 0.05 mm to about 500 mm, a particlewidth of about 0.03 mm to about 7 mm, and a particle height of about0.03 mm to about 1.0 mm, or alternatively in any subranges within theseranges. For example, at least 50%, or at least 60%, or at least 70%, orat least 80%, or at least 90% of the mass of the particles may have aparticle length of about 0.08 mm to about 100 mm, or 10 mm to about 70mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm toabout 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm;and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

For example, at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90% of the particles may have a particle length ofabout 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm toabout 200 mm; a particle width of about 0.05 mm to about 2 mm, or about1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle heightof about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, orabout 0.08 mm to about 1.0 mm.

Such particles mimic the texture and chewiness of meat products such aschicken nuggets or hamburgers, and are useful in the preparation of suchproducts, such as a filler or extender of meat products, or theirvegetarian versions. In the case of use of particles of the invention asa filler or extender of meat product, the ratio of filamentous fungalparticles to meat can range from 10:90 to 90:10 or any ratio in between.

For example, in some embodiments, the filamentous fungal particlescomprise particles having at least 90% of the particles with lengthsless than about 1.5 mm and the majority of lengths being 1 mm or less,widths of less than about 1 mm, and heights of less than about 0.75 mm.Food materials comprising such particles is characterized as having ahigher perceived density in the mouth, is easier to chew, offers acreamy mouth feel and a more refined food experience, and such particlesmay be used to prepare a food material that resembles a hamburger foundin fine dining establishments.

In some embodiments, the filamentous fungal particles comprise particleshaving at least about 90% of the particles with lengths between about 4mm and about 10 mm, widths of about 1.0 mm to about 3 mm, and heights ofless than 0.75 mm. Food materials comprising such particles is found tolead a more heartier food experience similar to the type of burgerprepared commonly found in burger restaurants or BBQ's.

Fine Particles (Flour)

In some embodiments, the inactivated edible filamentous fungal biomatsare reduced to fine particles. In some embodiments, the particles offilamentous fungus are in the form of a flour. In such embodiments, theparticle size and particle size distributions may be the same or similarto those conventional for flour-like materials, such as wheat or otherflours. In some embodiments, at least 50%, or at least 60%, or at least70%, or at least 80%, or at least 90% of the particles fall within therange of 0.03 mm to about 0.4 mm, or alternatively in any subrangewithin this range, such as about 0.03 mm to 0.07 mm, about 0.07 mm toabout 0.12 mm, about 0.12 mm to about 0.15 mm, about 0.15 mm to about2.0, about 0.04 mm to about 0.2 mm, or 0.06 mm to about 0.120 mm or 0.2mm to about 0.4 mm. In some embodiments, at least 50%, or at least 60%,or at least 70%, or at least 80%, or at least 90% of the particles fallwithin the range of 0.075 mm to about 0.12 mm.

In some embodiments, at least 50%, or at least 60%, or at least 70%, orat least 80%, or at least 90% of the mass of the particles fall withinthe range of 0.03 mm to about 0.4 mm, or alternatively in any subrangewithin this range, such as about 0.03 mm to 0.07 mm, about 0.07 mm toabout 0.12 mm, about 0.12 mm to about 0.15 mm, about 0.15 mm to about2.0, about 0.04 mm to about 0.2 mm, or 0.06 mm to about 0.120 mm or 0.2mm to about 0.4 mm. In some embodiments, at least 50%, or at least 60%,or at least 70%, or at least 80%, or at least 90% of the mass of theparticles fall within the range of 0.075 mm to about 0.12 mm.

The size reduction may be done using a flour mill, grinder or otherconventional equipment for size reduction.

In some embodiments, the moisture content of fine particulate materialof the invention is less than about 15%, about 14%, about 13%, about12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about5%, about 4%, about 3%, about 2% or about 1%. The low moisture levelsaid to prevent clumping of the particles.

Such particles are useful in the preparation of food materials such asbaked goods, including but not limited to bread, rolls, muffins, cakes,cookies, pies, etc. or can be sprinkled on other food products.

Liquid Dispersion (Milk)

One aspect of introducing protein into a foodstuff is to use a liquiddispersion made from the filamentous fungal biomat as a replacementingredient for milk or a milk analog. The liquid dispersion (alsoreferred herein as “milk”) comprises particles of filamentous fungalbiomats dispersed in an aqueous medium.

The size of the filamentous fungal biomat particles suitable for use inliquid dispersions is typically smaller than about 10 microns. In someembodiments, at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90% of the particles in a liquid dispersion fall withinthe range of about 1 microns to about 10 microns, or alternatively inany subrange within this range. In some embodiments, at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90% of theparticles are less than 10 microns, less than 9 microns, less than 8microns, less than 7 microns, less than 6 microns, less than 5 microns,less than 4 microns, less than 3 microns, less than 2 microns, or lessthan 1 micron. In some embodiments, at least about 50%, or at leastabout 60%, or at least about 70%, or at least about 80%, or at leastabout 90% of the particles may have a particle size of less than about 1micron.

The liquid dispersion or milk can be prepared by combining and blendinga filamentous fungal biomat with an aqueous phase, such as water. Theblended mixture can be heated gradually, such as to a boilingtemperature. The heated mixture is then allowed to cool. In someembodiments, a liquid dispersion can be produced under nitrogen. Thisprocess results in a creamier consistency of liquid dispersion with lessfungal scent. Production under nitrogen can be accomplished by bubblingwith nitrogen in a closed vessel such that nitrogen replaces most all ofthe available oxygen, either during blending, such as with a Vitamix orin a high-energy size reduction or milling process, or in the heatcycle. An exemplary method is described in Example 27.

The filamentous fungal biomat to water ratio can be adjusted to producea liquid dispersion of the appropriate consistency and density. Theratio of the biomat to water can range from about 1:10 to about 10:1 orany range of ratios in between. In some embodiments, the ratio of thebiomat to water can be about 1:10, about 1:9, about 1:8, about 1:7,about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1,about 9:1, about 10:1.

In various embodiments, a liquid dispersion of the invention is stablesuch that the particulates of filamentous fungus do not readily separatefrom the liquid medium in which they are dispersed. For example, uponforming the dispersion, the formed liquid appears to be homogeneous inappearance and does not visibly separate into distinct phases. Forexample, no visibly discernable or significant sediment forms on thebottom of the container holding the dispersion. In some embodiments, theliquid dispersion remains stable for at least about 1, 2, 3, 4, 5, 6, 9,12, 15,18, 21, or 24 hours or alternatively, it can remain stable for atleast about 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks, or 1,2, 3, 4, 5, or 6 months. In these embodiments, the dispersion can eitherbe at room temperature or at refrigerated temperatures, such as at about35° F. (1.6° C.).

Example 26 illustrates the stability of a liquid dispersion, i.e., amilk, of the invention. A milk that sat undisturbed in a refrigeratorfor 15 days and 30 days, and no visible separation was observed ineither sample. The milk also did not exhibit degradation of flavor orsmell.

In some embodiments, the dispersion comprises at least about 4%, atleast about 5%, at least about 6%, at least about 7%, at least about 8%,at least about 9%, at least about 10%, at least about 11%, at leastabout 12%, at least about 13%, at least about 14%, at least about 15%,at least about 16%, at least about 17%, at least about 18%, at leastabout 19%, at least about 20% solids. In other embodiments, a liquiddispersion of the invention will have a solids content of between about4% and about 30% or any sub-range between 4% and 30%.

The liquid dispersion can be used as a drink or beverage, including as asubstitute for any milk product such as dairy milk, almond milk, ricemilk, soy milk etc. It can be used in a number of recipes includingsoups, ice cream, yogurt, smoothies, fudge, and candies such as carameland truffles. In some cases, the filamentous fungal biomats producedfrom different feedstocks/carbon sources result in liquid dispersionshaving different flavors. For example, when the feedstock/carbon sourceis glycerol, the resulting liquid dispersion produced from Fusariumstrain MK7 is sweeter while a liquid dispersion resulting from Fusariumstrain MK7 grown on an acid whey feedstock/carbon source tends to besourer. The native sweetness or sourness of the filamentous fungus, e.g.Fusarium strain MK7, transfers to the ultimate food product. Forinstance, acid whey liquid dispersions lends itself to yogurt, whileglycerol liquid dispersions tends to lend itself to mouse, caramel orfudge.

In some embodiments, the liquid dispersion can be used to form a stablefoam, in that it forms a foam that does not collapse spontaneouslyimmediately upon cessation of the foaming process. The foaming processcan include whipping with a whipping appliance, incorporation ofcompressed gases or other conventional foaming processes. The foam issmooth and creamy in appearance and shows the presence of bubbles in adistribution of sizes. The larger bubbles tend to pop after sitting orbeing poured, but the smaller bubbles stay in suspension for a long timeto form a stable foam product. A foam product of the invention has thecompositional characteristics of a liquid dispersion and additionallyhas air or other gas incorporated into the foam in a stable manner. Forexample, a foamed material of the invention can have an increased volume(i.e., overrun) by incorporation of air of at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 75%, at least about 100%, at least about 200%, at leastabout 300%, at least about 400%, or at least about 500%, as compared tothe starting volume of the liquid dispersion prior to foaming. Invarious embodiments, a foamed material is stable for at least about 1day, at least about 2 days, at least about 3 days, at least about 4days, or at least about 5 days, at least about 6 days, at least about 7days, at least about 8 days, at least about 9 days, at least about 10days, at least about 11 days, at least about 12 days, at least about 13days, at least about 14 days, or at least about 15 days, at least about16 days, at least about 17 days, at least about 18 days, at least about19 days, at least about 20 days, at least about 21 days, at least about22 days, at least about 23 days, at least about 24 days, or at leastabout 25 days, at least about 26 days, at least about 27 days, at leastabout 28 days, at least about 29 days, at least about 30 days. In someembodiments, the liquid dispersion remains stable for at least about onemonth, at least about two months, or at least about three months. Asused in reference to a foam, stability refers to retaining at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95% of its initial foamed volume.

An average overrun of about 12% is suitable for preparing ice cream(with more fat and emulsifiers), frozen yogurt, cheesecake batters,whipped toppings etc. In some embodiments, the foam may incorporatenitrogen to provide different overrun characteristics.

In some embodiments, the food material is a cultured food product. Asused herein, unless otherwise specified, the term “cultured foodproduct” refers to a food product in which a microbial food culture,i.e. live bacteria, yeasts, or molds, is introduced to a filamentousfungus. By way of non-limiting example, fungal food materials accordingto the present invention may be cultured with Lactobacillus spp. orother lactic acid bacteria (to make, e.g., a yogurt analog food productor other dairy analog food product), Saccharomyces cerevisiae or otheryeasts used in brewing or baking (to make, e.g., a baked good analogfood product or an alcoholic beverage analog food product), moldstraditionally used to make sausages (e.g. Penicillium chyrsogenum orPenicillium nalgiovense, to make a sausage analog food product) or soysauces (such as Aspergillus oryzae or Aspergillus sojae, to make a soysauce analog food product), and so on. In some embodiments, culturedfood products according to the present invention may be cultured withtwo or more microbial food cultures, either simultaneously orsequentially, to produce an analog of a food product that is made byfermentation of two or more microbial cultures; by way of non-limitingexample, cultured food products according to the present invention mayinclude semi-soft ripened cheese analog food products (made bysubjecting a fungal material to a first culture by Lactobacillus spp. orother lactic acid bacteria and a second culture by a cheese ripeningyeast), blue cheese analog food products (made by subjecting a fungalmaterial to a first culture by Lactobacillus spp. or other lactic acidbacteria and a second culture by a mold such as Penicillium roqueforti),soft ripened cheese (e.g. Brie or Camembert) analog food products (madeby subj ecting a fungal material to a first culture by Lactobacillusspp. or other lactic acid bacteria and a second culture by Penicilliumcamemberti), etc.

In some embodiments, the food material comprises a yogurt analog foodproduct comprising the particles of the filamentous fungal biomats ofthe present invention dispersed in an aqueous medium. In someembodiments of the yogurt analog, the ratio of filamentous fungalparticles to water may range from about 1:10 to about 10:1. A higherratio of ratio of filamentous fungal particles to water is expected toincrease the texture and reduce runniness of the yogurt analog foodproduct. In some embodiments, the ratio of filamentous fungal particlesto water may be about 1:3, 1:2, 1:1 or 2:1.

In some embodiments, the yogurt analog food product comprises an invertsugar or inverted sugar. Invert sugar is resistant to crystallizationand promotes retention of moisture. and is used commercially in variousfoods such as baked goods, confections, or fruit preserves and beveragesto enhance flavor and texture and prolong shelf life. Examples includehoney or a mixture of glucose and fructose that is obtained byhydrolysis of sucrose and is sweeter than sucrose.

In some embodiments, the yogurt analog food product comprises athickening or gelling agent. Such agents are known in the art andinclude but are not limited to: agar, gelatin, starches (i.e. arrowroot,tapioca, corn, potato), higher fat liquids (coconut milk), fat (i.e.coconut flakes, deodorized or otherwise), chickpea water, flax seeds,xanthan gum, guar gum, psyllium husk, ground chia seed, nut/seedbutters, pumpkin puree, cooked mashed yams/sweet potato, applesauce,mashed overripe bananas or plantains, pureed dates or prunes, soaked andsimmered figs, shredded fruit/vegetables, shredded coconut, gluten freeflours (e.g. teff flour, buckwheat flour, amaranth flour, chickpeaflour, sorghum flour, almond flour), cooked pureed beans, cocoa Powder,vegetable gums, polysaccharides, vegetable mucilage, seaweedderivatives, pectin, gluten, soy and egg analogs. A thickening agent maybe a fat, which may be a liquid such as coconut milk, or a solid such asdeodorized coconut flakes.

In some embodiments, the cells of the filamentous fungi are lysed, whichreleases more protein and leads to increased thickening and potentiallygreater bioavailability of the nutrients. The lysis may be effected byany methods known in the art such as sonication.

In some embodiments, the yogurt analog food product comprises lacticacid bacteria (LAB). These bacteria produce lactic acid as the majormetabolic end product of carbohydrate fermentation. Examples of LABinclude the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus,and Streptococcus. In some embodiments, it comprises the bacteriaLactobacillus bulgaricus and/or Streptococcus thermophilus.

In some embodiments, the yogurt analog food product further comprises arennet. The rennet may be derived from an animal source, a vegetariansource or a microbial source. In vegetarian or vegan food products, therennet is derived from a vegetarian source and/or a microbial source.

In some embodiments, the yogurt analog food product further comprises anenzymatic water. For example, an enzymatic water can be produced asfollows. 100 gm of whole rye or durum wheat seeds (or other suitablewhole cereal seeds) may be combined with 1 liter of water, andgerminated for 2-4 hours. When seeds start to sprout and the first rootsappear, the seeds may be placed into a clean jar with 1 liter of water.The jar may be covered with a permeable cloth (linen or cotton), andincubated at room temp for 24 hours, at the end of which the water inthe jar changes color and odor. This water is referred to as enzymaticwater and can be used in the production of yogurt and cheese.

In some embodiments, the yogurt analog food product further comprises aprobiotic. Probiotics are mixtures of live micro-organisms such asbacteria and yeast that provide health benefits including improveddigestion.

In some embodiments, the yogurt analog food product comprises milksolids derived from animal milk. In some embodiments, the yogurt analogfood product is free of milk solids derived from animal milk, i.e. itdoes not contain any milk solids.

It is to be expressly understood that in addition to yogurt analog foodproducts, food materials of the present invention may comprise any oneor more other dairy analog food products. By way of first non-limitingexample, food materials of the present invention may comprise a cheeseanalog food product, such as a hard cheese (e.g. Parmesan) analog foodproduct, a semi-hard cheese (e.g. Gouda) analog food product, asemi-soft cheese (e.g. Havarti) analog food product, a soft or softripened cheese (e.g. Brie) analog food product, a cream cheese analogfood product, a sour milk cheese analog food product, a blue cheeseanalog food product, a mascarpone cheese analog food product, a pastafilata (e.g. Mozzarella) cheese analog food product, a brined cheese(e.g. Feta) analog food product, a whey cheese (e.g. Ricotta or Brunost)analog food product, or a fresh cheese (e.g. cottage cheese) analog foodproduct. By way of second non-limiting example, food materials of thepresent invention may comprise a butter analog food product, such as araw cream butter analog food product, a butterfat analog food product, aclarified butter analog food product, a whey butter analog food product,a cultured butter analog food product, a mild cultured butter analogfood product, a sweet cream butter analog food product, or a traditionalbuttermilk analog food product. By way of third non-limiting example,food materials of the present invention may comprise a whey analog foodproduct, such as a sour whey analog food product or a sweet whey analogfood product. By way of fourth non-limiting example, food materials ofthe present invention may comprise a cream analog food product, such asa crème fraiche analog food product, a smetana analog food product, asour cream analog food product, a half-and-half analog food product, atable cream analog food product, a whipping cream analog food product, adouble cream analog food product, a clotted cream analog food product, asoured cream analog food product, a pasteurized cream analog foodproduct, or a condensed cream analog food product. By way of fifthnon-limiting example, food materials of the present invention maycomprise a sour milk analog food product, such as a quark analog foodproduct, a cheese curd analog food product, a soured milk analog foodproduct, a kefir analog food product, an organic yogurt or mild yogurtanalog food product, a yogurt analog food product, a cream yogurt analogfood product, or a cultured buttermilk analog food product. By way ofsixth non-limiting example, food materials of the present invention maycomprise a milk analog food product, such as a raw milk analog foodproduct, a lowfat or skimmed raw milk analog food product, a pasteurizedmilk analog food product, a fresh whole milk analog food product, alowfat milk analog food product, a skimmed milk analog food product, anextended shelf-life (ESL) milk analog food product, an ultra-hightemperature processed (UHT) milk analog food product, a sterilized milkanalog food product, a condensed or evaporated milk analog food product,a part-skim condensed milk analog food product, or a condensed skimmedmilk analog food product. By way of seventh non-limiting example, foodmaterials of the present invention may comprise a powdered dairy analogfood product, such as a powdered whey analog food product, a powderedmilk analog food product, or a powdered skimmed milk analog foodproduct. Dairy analog food products according to the present inventionmay, in embodiments, be vegan food products, i.e. food products thatcontain no animal products, and thus allow observers of vegan diets toincorporate such dairy analogs into their diet.

Particles of the filamentous fungal biomat can be added as a protein orother nutritional source to augment the nutritional content of afoodstuff or can be, for example, the sole protein component. For foodscomposed entirely of filamentous fungal biomats, or the size-reducedparticles of such biomats, the particles can be optimized for particulartextures, mouthfeel, and chewiness. The ability to alter texture, mouthfeel, and chewiness allow customization to accommodate individualshaving particular dietary needs, such as those that have troublechewing, or who require/desire softer foods while still providing thesame nutritional and taste experience or those who desired food withmore texture, more mouthfeel and more mastication. Because of theability to easily control the particle size, foods augmented withfilamentous fungal biomats or made solely from filamentous fungalbiomats have textures very similar to the standard protein foods thatthey emulate, as can be seen in Table 2.

TABLE 2 Results from Stable Micro Systems TA XT plus texture analyzerAvg. Max Avg. Area Avg. Mean Food Hardness (g/mm) (g) Parameters FishStick Pre-Test Speed: 2.00 mm/sec Commercial  3654 ± 1774 17868 ± 5674 894 ± 284 Test Speed: 4.00 mm/sec fish stick Post-Test Speed: 10.00mm/sec MK7 fish stick 1618 ± 180 19990 ± 610  1000 ± 100 Target Mode:Distance Chicken Nugget Force: 100.0 g Commercial  3838 ± 56.8 27329 ±3663 1367 ± 183 Distance: 20.000 mm chicken nugget Strain: 10.0% Quornchicken   4013 ± 1066.3  27751 ± 1346.4  1415 ± 111.4 Trigger Type: Auto(Force) nugget Tigger Force: 5.0 g MK7 small  3127 ± 19.7 33065 ± 34581654 ± 173 Probe: HDP/WBV particle Warner Bratzler V Slot Blade MK7medium 2514 ± 663 27217 ± 6437 1361 ± 322 particle MK7 large  3461 ±77.8  34591 ± 2971.2  1730 ± 14.6 particle Burger 100% Beef 4326 ± 71412350 ± 46.1   1727 ± 14.1 burger 90% Beef, 10% 5011    14048 1929 MK780% Beef, 20% 2615 ± 199 10641 ± 511  1456 ± 46  MK7 70% Beef, 30% 2240± 262  9859 ± 2947 1291 ± 300 MK7 60% Beef, 40% 2094 ± 156  8118 ± 10881155 ± 180 MK7 100% MK7,  2228 ± 1988 5079 ± 964  1089 ± 70.6 chopped(highly processed) Firmness Food (g) Chocolate Pre-Test Speed: 1.00mm/sec Mousse Test Speed: 1.00 mm/sec Nestle 182.45 Post-Test Speed:10.00 mm/sec chocolate Target Mode: Distance mousse T.A. Variable No: 5:0.0 g MK7 chocolate 135.09 Distance: 10.000 mm mousse Strain: 10.0%Trigger Type: Auto (Force) Tigger Force: 5.0 g Probe: P/25; 25 mm DIACylinder Aluminum

Particles of the filamentous fungal biomats can be used as sole proteincomponents in a food material or can be used to augment protein contentof other food materials. Examples of foods that can be produced usingonly the reduced particle size of the filamentous fungal biomat, with orwithout added flavorings, include without limitation meat-likevegetarian or vegan products (e.g., ground beef, ground chicken, groundturkey, chicken nuggets, fish sticks or patties, jerky), snacks (e.g.chips), soups, smoothies, beverages, milk analogs, breads, pastas,noodles, dumplings, pastries (e.g. Pate a Choux), cookies, cakes, pies,desserts, frozen desserts, ice cream analogues, yogurt, confections, andcandy.

Foods augmented with the reduced particle size of the filamentous fungalbiomat can significantly increase the protein content, which isparticularly important for individuals following a vegan diet. Forexample, soups, drinks or smoothies can be augmented with strain MK7liquid dispersion.

Whether biomat particles of reduced size are used to augment the proteincontent of food or is used as the sole protein component, in someinstances binders are helpful in achieving the desired texture. Approvedfoodstuff binders are suitable, such as egg albumen, gluten, chickpeaflour, vegetarian binders, arrowroot, gelatin, pectin, guar gum,carrageenan, xanthan gum, whey, chick pea water, ground flax seeds, eggreplacer, flour, agar-agar, Chia seeds, psyllium, etc. which can be usedsingularly or in combination. In addition to foodstuff binders, thereduced particle size of the filamentous fungal biomat can also be mixedwith approved flavors, spices, flavor enhancers, fats, fat replacers,preservatives, sweeteners, color additives, nutrients, emulsifiers,stabilizers, thickeners, pH control agents, acidulants, leaveningagents, anti-caking agents, humectants, yeast nutrients, doughstrengtheners, dough conditioners, firming agents, enzyme preparations,gasses, and combinations thereof. Typically, binders, flavors, spices,etc. are selected to meet the demands of a particular population. Forexample, milk and/or milk solids are not used to accommodate individualswith dairy allergies/sensitivities, wheat flour may not be used toaccommodate those with gluten allergies/sensitivities, etc.

In some applications, a substantially unimodal particle sizedistribution, i.e. in which all particles are approximately the samesize, may be used, while in other applications a broad or multimodaldistribution or combination of distributions of particle size may beused. Similarly, size-reduced particles can be derived from a singlesource of filamentous fungal biomat or from a combination of differentsources of filamentous fungal biomats; e.g. strain MK7 alone or strainMK7 and Fusarium venenatum, or strain MK7 and Fusarium venenatum andGiant Puffball biomats, etc.

The use of filamentous fungi for commercial production in the past hasgenerally required significant infrastructure and/or equipment, energyrequirements, expensive reagents, and/or significant human resources.Filamentous fungi are well known for having the greatest metabolicdiversity of all microorganisms on Earth, including the ability toproduce a wide spectrum of organic acids, antibiotics, enzymes,hormones, lipids, mycotoxins, vitamins, organic acids, pigments, andrecombinant heterologous proteins (Wiebi (2002) Myco-protein fromFusarium venenatum: a well-established product for human consumption.Appl Microbiol Biotechnol 58, 421-427; El-Enshasy (2007) Chapter9—Filamentous Fungal Cultures—Process Characteristics, Products, andApplications. In. Bioprocessing for Value-Added Products from RenewableResources. Editor: Shang-Tian Yang. Elsevier; Gibbs et al (2000) Growthof filamentous fungi in submerged culture: problems and possiblesolutions. Crit. Rev. Biotechnol. 20, 17-48), as well as the ability todegrade many types of recalcitrant materials such as lignocellulose andhumic substances in soils.

While widely used, significant challenges to production by submergedfermentation still exist and include important factors such as growthlimitation due to the restricted oxygen availability and excessive shearforces generated by agitation (Gibbs et al (2000) Growth of filamentousfungi in submerged culture: problems and possible solutions. Crit. Rev.Biotechnol. 20, 17-48). Since oxygen solubility in water under Earthsurface conditions is about 8 mg/L, it is readily depleted during rapidgrowth in submerged cultures. Thus, continuous aeration using complex,expensive and energy intensive aeration and agitation systems isrequired to maintain high growth rates. The cultivation of filamentousfungi is even more challenging since the filamentous morphology impartsnon-Newtonian rheological behavior that further inhibits oxygen transferto solution (Nørregaard et al. (2014) Filamentous Fungi Fermentation. InIndustrial Scale Suspension Culture of Living Cells, H.-P. Meyer, andD.R. Schmidhalter, eds. (Wiley-VCH Verlag GmbH & Co. kGaA), pp.130-162). As culture densities increase, the amount of energy requiredto aerate and mix the cultures increases nonlinearly as well as theenergy requirements to aerate dense cultures are very high. For manyfilamentous species, vigorous agitation and aeration of the culturesbecomes detrimental to hyphal growth and as a result dramaticallydecreases growth rate. These and other challenges to submergedfermentation of filamentous microorganisms require innovative solutionsto effectively harness these organisms with the limited resourcesavailable in spacecraft and at extraterrestrial stations.

The disclosed reactor system (1) addresses these problems and has thefollowing advantages:

-   -   Active aeration or agitation of the liquid culture is not        necessary;    -   In-situ aggregation of biomass into a single coherent mat with        significant tensile strength (>0.1 kg/cm of biomat width) which        allows easy harvesting;    -   Textured biomats can be used for a wide variety of mission        critical products (i.e. food, bioplastics, biofuels, nutritional        supplements, and as an expression platform for a variety of        pharmaceuticals;    -   Minimal water use as well as minimal and/or no residual waste        water or nutrients from the process while maintaining high        biomass production (80-120 g/m²/d or 0.55 g/L/h)    -   Growth rates can translate to the production of fully formed        biomats in as little as 2 days or can be further expanded for        more than 10 days, in some embodiments up to at least about 21        days;    -   High biomass density (biomats are typically 0.684 g/cm³ wet        weight or 0.123 g/cm³ dry weight);    -   High yield (6.9 kg/m² wet weight or 1.23 Kg/m² dry weight total,        and in some embodiments up to at least about 206 g/m² dry weight        per day);    -   A variety of filamentous fungi (including extremophiles, as well        as known edible and commercially relevant mushrooms) with        specific advantages for different processes can be grown;    -   Scale-up or down is relatively straightforward and does not        result in decreased productivityup to a growth area of at least        about 150 cm²;    -   Process can use a very wide variety of C and N-rich waste        substrates that arise from natural disasters and/or space        missions.

The disclosed reactor system provides a self-contained biomat reactorcomprising a container and placed within the container a feedstock, afungal inoculum, an at least semi-permeable membrane (e.g.gas-permeable, liquid-permeable, gas-semi-permeable, and/orliquid-semi-permeable), and optionally a liquid nutrient medium.Depending upon the circumstances, the reactor can be a one-time usereactor or a reusable reactor.

Typically, the container is capable of being sealed and may include acontainer cover in addition to a seal. Some container examples are acovered tray, a covered petrie dish, another type of covered container,or a bag. For some uses or in some environments the container has aplurality of growth chambers, for example following a manifold designand/or a baffling system.

The feedstock is inoculated with a filamentous fungal strain asdescribed above. Examples of Ascomycetes strains are strain MK7 (ATCCPTA-10698 deposited with the American Type Culture Collection, 1081University Boulevard, Manassas, Va., USA), Fusarium venenatum, Fusariumavenaceum, and/or combinations thereof. Inoculation of the feedstock canoccur at the time the feedstock is placed within the container or canoccur sometime after the feedstock has been placed. That is, the reactor(1) can be primed with freeze-dried filamentous fungal inoculum that isrevived upon contact with the feedstock or the feedstock can be directlyinoculated after placement in the reactor channel(s) (4) or thefeedstock can be inoculated and then placed in the reactor channel(s).

With respect to the feedstock used in the reactor, the feedstock can beas described above. For example, it can be a waste product, such asnaturally occurring urine and/or feces, food waste, plant material,industrial waste such as glycerol, and waste by-products, starch and/orby products of starch hydrolysis, acid whey, sugar alcohol, and/orcombinations thereof. Synthesized or manufactured waste surrogates, suchas surrogate human urine can also be used. Plant material feedstocks aretypically lignocellulosic. Some examples of lignocellulosic feedstockare agricultural crop residues (e.g. wheat straw, barley straw, ricestraw, small grain straw, corn stover, corn fibers (e.g. corn fiber gum(CFG), distillers dried grains (DDG), corn gluten mean (CGM), switchgrass, sugar beet pulp, waste streams from palm oil production,hay-alfalfa, sugarcane bagasse, non-agricultural biomass (e.g. algalbiomass, cyanobacterial biomass, urban tree residue), forest productsand industry residues (e.g., softwood first/secondary mill residue, hardsoftwood first/secondary mill residue, recycled paper pulp sludge),lignocellulosic containing waste (e.g. newsprint, waste paper, brewinggrains, used rubber tire (URT), municipal organic waste and by-products,yard waste and by-products, clinical organic waste and by-products, andwaste and by-products generated during the production of biofuels (e.g.processed algal biomass, glycerol), and combinations thereof.

In embodiments, the permeable membrane may comprise a polymericmaterial, such as, by way of non-limiting example, a polypropylene, apolyethylene, a polytetrafluoroethylene, a polycarbonate, a polyamide(e.g. nylon), a polypyrrolone, a poly(amidoamine) dendrimer composite, apolyvinylidene fluoride, a polyethersulfone, cellulose acetate, a mix ofcellulose esters, and/or butadiene-acrylonitrile. The permeable membranemay comprise, additionally and/or alternatively, a glass fiber material,a porous ceramic material, and/or a fabric, such as, by way ofnon-limiting example, a polypropylene fabric, a polytetrafluoroethylenefabric, and/or a nylon net filter. While the pore size of the permeablemembrane (2) typically may be any 0.01 μm increment value or range ofvalues between about 0.2 μm and about 25 μm, including, by way ofnon-limiting example, 0.2 μm, 0.22 μm, 0.45 μm, 1.0 μm, 5 μm, 10 μm, and11 μm, the membrane (2) can be in the form of a sterile cloth-likematerial or the form of a paper-like material.

A permeable membrane(s) (2) allows optimization of the system in severaldifferent ways that are illustrated in FIGS. 15-18. While the reactorsystem illustrated in the Figures has a total of nine channels (4), theskilled artisan appreciates that any number of channels (4) can bepresent, from a single channel (4) to a plethora of channels (4),depending on the space available for placement the reactor (1).Similarly, the shape of the channels (4) is not limited to a rectangularprisms or cylinders and can take any shape suitable to fit the spaceavailable for the reactor (1).

In some cases, the membrane (2) is placed in direct contact with thesurface of the feedstock, optional liquid media, and inoculum present inthe container as shown in FIG. 12. The membrane can also be sealed incontact with the surface of the feedstock, for example, by attaching itto a plastic frame with an integrated rubber gasket.

In other instances, the membrane is suspended over the feedstock so thatas the fungi grows and consumes oxygen, the membrane drops down towardsthe mat or onto a baffle system located between the membrane and thefeedstock which allow for growth of aerial hyphae. Such a system isshown in FIG. 15. Here, the reactor (1) is comprised of multiplechannels (4) which initiate at an inlet valve (6) at the front (7) ofthe reactor, terminate at an outlet valve (8) at the back (5) of thereactor, and are separated by baffles/walls (9). A gas permeablemembrane (2) forms the top of the reactor. The bottom (3) of the reactorcan be formed of any suitable substance including, but not limited toboth hard and soft plastics such as polyethylene terephthalate, highdensity polyethylene, polyvinyl chloride, polyactic acid, polycarbonate,acrylic, acetal, nylon, acrylonitrile butadiene styrene, glass, metalssuch as aluminum, titanium, stainless steel etc. and/or combinationsthereof. The baffles/walls (9) can be made of similar materials.Suitable front (6) and back (8) valves include, but are not limited to,one-way valves, 2-way valves, ball valves, butterfly valves, gatevalves, plug valves, globe valves, pinch valves, disc check valves,attached valves, detached valves, and/or combinations thereof. The inletvalve (6) serves to provide access to the chamber (4) for delivery offeedstock/media to the chamber while the outlet valve (8) allows removalof exhausted feedstock and/or filamentous fungal biomat. The permeablemembrane (2) may comprise a polymeric material, such as, by way ofnon-limiting example, a polypropylene, a polyethylene, apolytetrafluoroethylene, a polycarbonate, a polyamide (e.g. nylon), apolypyrrolone, a poly(amidoamine) dendrimer composite, a polyvinylidenefluoride, a polyethersulfone, cellulose acetate, a mix of celluloseesters, and/or butadiene-acrylonitrile. The permeable membrane maycomprise, additionally and/or alternatively, a glass fiber material, aporous ceramic material, and/or a fabric, such as, by way ofnon-limiting example, a polypropylene fabric, a polytetrafluoroethylenefabric, and/or a nylon net filter. While the pore size of the permeablemembrane (2) typically may be any 0.01 μm increment value or range ofvalues between about 0.2 μm and about 25 μm, including, by way ofnon-limiting example, 0.2 μm, 0.22 μm, 0.45 μm, 1.0 μm, 5 μm, 10 μm, and11 μm, the membrane (2) can be in the form of a sterile cloth-likematerial or the form of a paper-like material. For some uses, themembrane's surface is smooth in texture, for others the surface is roughin texture. In addition, the path for gas diffusion can vary from beingessentially direct to following a more tortuous path.

In other situations, the membrane facilitates ingress of oxygen andegress of other gases produced during fungal growth (FIG. 14). In thissituation the hermetic reactor (1) has a gas collection chamber (14)that is immediately atop of the gas permeable membrane (2) (see FIG.16). The gas collection chamber (14) can be made of similar materials tothose used for the walls/baffles (9) or the bottom (3) of the reactor;i.e. both hard and soft plastics such as polyethylene terephthalate,high density polyethylene, polyvinyl chloride, polylactic acid,polycarbonate, acrylic, acetal, nylon, acrylonitrile butadiene styrene,glass, metals such as aluminum, titanium, stainless steel etc. and/orcombinations thereof. Alternatively, the gas collection chamber iscomprised of channels (15) which can mirror the channels (4) of thehermetic reactor (1) or which encompass more than one of the hermeticreactor channels (20) (see FIG. 17).

In yet other systems, separate gas permeable membranes are used foringress and egress of gases. FIG. 18 illustrates such a system. In thisinstance, two different gas permeable membranes (2, 50) feed intoseparate gas collection channels (30, 40) and are present over a singlereactor channel (4). This type of system allows ingress, egress, and/orcollection and/or separation of distinct useful gases. As an example,one membrane might be calibrated for oxygen passage and the secondmembrane calibrated for carbon dioxide or hydrogen passage or otherrelevant gas systems.

The filamentous fungus may be inoculated, and the biomat may be grown,on either or any side of the membrane, including, by way of non-limitingexample, an upper side, a lower side, an atmosphere-facing and/or gasheadspace-facing side, or a feedstock-facing side. Biomat growthcharacteristics, including but not limited to density and the side ofthe membrane on which the biomat grows, may be controlled by controllingvarious parameters of the bioreactor, e.g. the side of the membrane onwhich the fungus is inoculated, membrane material, membrane pore sizeand thickness, temperature, humidity, pressure, wavelength or amount oflight and so on. It has generally been found that the best quality ofbiomat is achieved when the feedstock is constantly in interaction (i.e.physical contact) with the permeable membrane, and the biomat grows onthe opposite (i.e. atmosphere- or gas headspace-facing) side of themembrane. Particularly, it may in some embodiments be advantageous forthe biomat to grow into a gas headspace that is sealed or otherwiseisolated from a surrounding atmosphere and is allowed to become humid asthe biomat grows and produces water. It is also advantageous for thebioreactor to be configured to allow the biomat, and in particularhyphae of the filamentous fungus of the biomat, to remain substantiallydry (i.e. not in contact with liquid growth medium or other free liquidmoisture) during biomat growth.

As the biomat grows, the respiration process of the growing fungusproduces water, which accumulates within the bioreactor (generally onthe gas phase side of the membrane). In addition to providing a humidenvironment suitable for further biomat growth, this accumulated watercan be collected as a valuable product in its own right and used for anysuitable purpose. Thus, one advantage of the present invention is that aportion of the feedstock, which can comprise a waste product such as thefeces or urine of an animal, may be converted into clean water. Otherfeedstocks from which clean water may suitably be produced may comprise,without limitation, any one or more of a sugar (e.g. sucrose, maltose,glucose, fructose, rare sugars, etc.), a sugar alcohol (e.g. glycerol,polyol, etc.), a starch (e.g. corn starch, etc.), a starch derivative, astarch hydrolysate, a hydrogenated starch hydrolysate, a lignocellulosicpulp or feedstock (e.g. sugar beet pulp, agricultural pulp, lumber pulp,distiller dry grains, brewery waste, etc.), corn steep liquor, acidwhey, sweet whey, milk serum, wheat steep liquor, industrial liquor,food refinery products/waste streams, agricultural crop residues (e.g.wheat straw, barley straw, rice straw, pea, oat, small grain straw, cornstover, corn fibers (e.g. corn fiber gum (CFG), distillers dried grains(DDG), corn gluten meal (CGM), switch grass, hay-alfalfa, sugarcanebagasse, non-agricultural biomass (e.g. algal biomass, cyanobacterialbiomass, urban tree residue), vegetables (e.g. carrots, broccoli,garlic, potato, beets, cauliflower), forest products and industryresidues (e.g., softwood first/secondary mill residue, hard softwoodfirst/secondary mill residue, recycled paper pulp sludge, anaerobicdigestate), lignocellulosic containing waste (e.g. newsprint, wastepaper, brewing grains, used rubber tire (URT), municipal organic waste,yard waste, clinical organic waste, sugar, starch, waste oils, oliveoils, olive oil processing waste, cricket excrement, and waste generatedduring the production of biofuels (e.g. processed algal biomass,glycerol).

As the biomat grows, the respiration process of the growing fungus mayalso produce one or more gases, e.g. ammonia, hydrogen gas, and/orvolatile esters. In embodiments, the membrane may be a selectivegas-permeable membrane that allows for separation and/or segregation ofat least one gas produced by fungal respiration.

Differences in pressure between one side of the membrane and the otherside of the membrane may be leveraged to affect various properties ofthe filamentous fungus of the biomat. For example, the inventioncontemplates non-atmospheric pressure on one or both sides of themembrane, such as super- or sub-atmospheric pressure on one side andatmospheric on the other or super-atmospheric or sub-atmospheric on bothsides or super-atmospheric on one side and sub-atmospheric on the other.By way of non-limiting example, the growth rate of the biomat may beaffected, e.g. a positive pressure on the feedstock side of the membranemay be similar to “force-feeding” the biomass and accelerating biomatproduction and/or increasing yield, while positive pressure on the “dry”side of the membrane may result in slower production of the biomass,resulting in an altered composition or physical property of the fungalbiomass. Conversely, in some embodiments, it may be advantageous forgrowth rate for the biomat to remain dry during growth, and thus theopposite effect may be observed, i.e. positive pressure results in a“wetter,” slower-growing mat while negative pressure results in a“drier,” faster-growing mat. Selective application of pressure may alsobe suitable to determine the type of membrane that is most advantageousfor a given application of the bioreactor, e.g. variations in pressuremay be more or less advantageous for membranes of a given pore size).Changes in atmospheric pressure in the gas headspace and/or environmentmay also affect biomat growth characteristics, e.g. aerial hyphaeproduction, production of hyphae, mycelia, and/or filaments, and biomassdensity, and may even be used to promote inoculation of the filamentousfungus on the membrane.

In embodiments, cyanobacteria may advantageously be provided on eitherside of the membrane within the bioreactor, either naturally or byinoculation. Cyanobacteria, whether living or dead, may advantageouslycontribute to the nutritive characteristics of the feedstock as a sourceof carbon and nitrogen. Additionally, living cyanobacteria may, as aproduct of respiration, serve as an oxygen source to promote the growthof the biomass. Either or both of these advantages may make thebioreactors of the present disclosure particularly suitable for variousapplications where other solutions are inviable, e.g. long-durationcrewed space missions. An exemplary schematic of an embodiment of abioreactor including respiring cyanobacteria is illustrated in FIG. 29.

While in most embodiments a gentle force must be applied to harvest(i.e. remove from the membrane surface) the biomats, in some embodimentsthe biomats can be “self-harvesting,” i.e., they can separate from themembrane spontaneously. The present inventors have particularly observedthis phenomenon, e.g., when the biomat has a high water content, whenoleic acid is present in the feedstock, and/or when the membrane isrelatively thin (e.g. less than 0.2 mm thick), smooth, and characterizedby low tortuosity. In some embodiments, self-harvesting may be achievedby application of a pressure. The adhesion strength of the biomat to themembrane may depend, and thus be selected by controlling, any one ormore bioreactor parameters, e.g. filamentous fungus species, feedstockcomposition, membrane characteristics (such as material, pore size,geometry/roughness, etc.), pressure on either side of the membraneand/or pressure differential across the membrane, and so on. It is thusone aspect of the disclosure to provide mats that harvest themselves byseparating from the membrane surface spontaneously.

The reactor (1) produces a biomat that serves as a food source, such asa protein source and/or an oil source. However, the biomat can alsoserve as a leather analog, a bioplastic, a source of biofuel precursors,a biofuel, and/or combinations thereof. In yet other embodiments, thebiomat serves to produce organic products such as organic acids,antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins, pigmentsand recombinant heterologous proteins.

The disclosed biomat reactor fermentation technology enables growth onstandard as well as extreme feedstocks and media, such as human waste(urine/feces), and produces a highly consolidated and textured productwithout the requirement of a separation or concentration step.Relatively high biomass production rates—i.e. at least about 0.05 g/L/hdry biomass (i.e., grams of dry biomass produced per liter of feedstockper hour), or at least about 0.10 g/L/h dry biomass, or at least about0.15 g/L/h dry biomass, or at least about 0.20 g/L/h dry biomass, or atleast about 0.25 g/L/h dry biomass, or at least about 0.30 g/L/h drybiomass, or at least about 0.35 g/L/h dry biomass, or at least about0.40 g/L/h dry biomass, or at least about 0.45 g/L/h dry biomass, or atleast about 0.50 g/L/h dry biomass, or at least about 0.55 g/L/h drybiomass, or at least about 0.60 g/L/h dry biomass, or at least about0.65 g/L/h dry biomass, or at least about 0.70 g/L/h dry biomass, or atleast about 0.75 g/L/h dry biomass, or at least about 0.80 g/L/h drybiomass, or at least about 0.85 g/L/h dry biomass, or at least about0.90 g/L/h dry biomass, or at least about 0.95 g/L/h dry biomass, or atleast about 1.00 g/L/h dry biomass—and/or for example, in the core ofbatch processes high production (i.e., grams of biomat produced perliter of feedstock) of at least about 10 g/L, or at least about 20 g/L,or at least about 30 g/L, or at least about 40 g/L, or at least about 50g/L, or at least about 60 g/L, or at least about 70 g/L, or at leastabout 80 g/L, or at least about 90 g/L, or at least about 100 g/L, or atleast about 110 g/L, or at least about 120 g/L, or at least about 130g/L, or at least about 140 g/L, or at least about 150 g/L, or at leastabout 160 g/L, or at least about 170 g/L, or at least about 180 g/L, orat least about 190 g/L, or at least about 200 g/L, are achieved withoutthe need for active aeration or agitation. Scale-up of the systemvertically, horizontally, and/or in more than two dimensions is simpleand does not result in decreased productivity. The produced biomats aretypically 0.2 to 2.5 cm thick with a dry matter content of 10-30% andcan be readily used for mission critical needs such as meatalternatives, a myriad of other appetizing foods, and buildingmaterials.

The fungal biomats grown in the disclosed reactor system can bedescribed as thick pellicles, which in many ways are similar to themicrobial biofilms that grow on surfaces, but are consistent withbiomats thicknesses described herein and are present at the gas-liquidinterface. For example, bacterial cells within biofilms have been shownto withstand extreme disinfection treatments with sodium hypochlorite(bleach) and sodium hydroxide (Corcoran, 2013). The disclosed reactorsystem takes advantage of the biofilm structure, enabling growth onharsh human and industrial wastes and by-products that may be generatedunder extreme conditions such as those generated on space missions or byother harsh conditions caused by natural disasters.

The disclosed reactor design incorporates a permeable membrane that sitsdirectly on or suspended just above the liquid surface of a feedstock.In one embodiment, encapsulated reactor design allows for gas exchangewith the exterior atmosphere but is hermetically sealed to keepcontaminants from entering or gases/liquids from escaping. Theencapsulated reactor design can also enable separation of consumablegases from evolved gases by way of gas permeable membrane. To accomplishthis, in some instances valves and/or additional porous membranes havingthe same or different properties are used to form distinct layersbetween various aspects of the one or more feedstocks and optionalliquid culture media.

Rapid biomat growth using the disclosed reactor design has beendemonstrated with a variety of permeable membrane materials. FIG. 13shows an approximately 7 mm thick biomat grown in reactor where thecontainer was a Petri dish covered with a polypropylene membrane whichwas laid directly on the feedstock/liquid medium surface. The initialbiomat formed by direct attachment to the membrane and grew downwardinto the liquid medium over time (see FIG. 12). By the end of a five-daygrowth period, essentially all of the feedstock/liquid medium wasconsumed and dense biomass completely filled the volume underneath themembrane.

The biomat produced only mildly adheres to the membrane and was easilyharvested by simply peeling away the biomat from the membrane (see FIGS.13A-13D). Additional experiments with polycarbonate membranes haveproduced similar results (data not shown). Thus, the total reactorvolume can be efficiently utilized to produce dense, easily harvestedbiomass. By way of non-limiting example, bioreactors of the presentinvention can take the form of a sealed “envelope,” comprising fourmembranes: an outer upper membrane, an inner upper membrane, an innerlower membrane, and an outer lower membrane. In such embodiments,feedstock may be provided between the inner upper membrane and the innerlower membrane, whereby the feedstock is “sandwiched” between gasheadspaces on either vertical side (i.e. between the outer and innerupper membranes and between the outer and inner lower membranes). Biomatcan thus grow on the outer surfaces of the inner membranes, into the gasheadspace on either side of the feedstock, while still being at leastpartially isolated from a surrounding atmosphere due to the outermembranes (which may in embodiments be gas-permeable orgas-semi-permeable membranes). The “envelope” bioreactor may be a highlyvolume-efficient bioreactor that may be used when space is at a premium,e.g. aboard a spacecraft or in emergency and rescue situations.

The biomats commonly produced in the disclosed reactors are highlydense, as described herein, and, depending on the fungus and growthconditions, exhibit a fibrous texture. Production of a fibrous biomasscan be crucial for certain mission critical products such as foods thatrequire texture to simulate meat, as well as fibrous materials thatsimulate leather and wood. The dense nature of the biomass also enableseasy harvesting without the need for a concentration step (e.g.,centrifugation, filtration). The density of the biomats can range fromabout 0.01 g dry biomat weight/cm³ to about 1 g/cm³, and any subrangewithin this range. In some embodiments, the density can be greater thanabout 0.01, greater than about 0.02, greater than about 0.03, greaterthan about 0.04, greater than about 0.05, greater than about 0.06,greater than about 0.07, greater than about 0.08, greater than about0.09, greater than about 0.1, greater than about 0.2, greater than about0.3, greater than about 0.4, greater than about 0.5, greater than about0.6, greater than about 0.7, greater than about 0.8, greater than about0.9, or greater than about 1 g/cm³.

Referring now to FIG. 23, several different bioreactor configurationsare illustrated, each of which is within the scope of the presentdisclosure. In the configuration labeled 1, the feedstock is disposedbelow and in physical contact with the membrane, and the biomat growsupwardly from the upper surface of the membrane (although in someembodiments, fungal material may also grow downwardly from the lower,i.e. feedstock-side, surface of the membrane). In the configurationlabeled 2, the biomat grows directly on the surface of the feedstock(i.e. without a membrane present between the biomat and the feedstock)into a gas headspace, and the feedstock, biomat, and headspace areseparated from a surrounding environment by a membrane; membranes ofthis embodiment may be similar to, or different from, the membranedisposed between the feedstock and the biomat in other embodiments. Inthe configuration labeled 3, two membranes are provided: a lowermembrane separating the feedstock from the biomat (as in theconfiguration labeled 1) and an upper membrane separating the biomat andgas headspace above the biomat from a surrounding environment (as in theconfiguration labeled 2). In the configuration labeled 4, an“upside-down” bioreactor scheme is provided, wherein the feedstock isprovided on the upper surface of the membrane and the biomat growsdownwardly from the lower surface of the membrane into a gas headspaceand/or the surrounding environment (although in some embodiments, fungalmaterial may also grow upwardly from the upper, i.e. feedstock-side,surface of the membrane).

Referring now to FIGS. 24A and 24B, a “hermetic” (tightly sealed againstthe environment) embodiment of a bioreactor according to theconfiguration labeled “1” in FIG. 23 is illustrated, before (FIG. 24A)and after (FIG. 24B) production of the biomat. As FIG. 24B illustrates,fresh water produced by respiration of the filamentous fungus hascondensed and accumulated on the gas-phase side of the membrane.

Referring now to FIG. 33, a nylon net filter membrane is illustrated. Inthis embodiment, the nylon net filter consists of two nylon membranes(pore size 11 μm) that are “stacked” (i.e. placed in physical contactsuch that a second surface of the first membrane abuts a first surfaceof the second membrane) to reduce the effective porosity of the membraneand allow the membrane to remain above the surface of the feedstock.Membranes of this type are suitable for growing fungal biomats,according to the present disclosure.

Referring now to FIGS. 35A and 35B, a membrane envelope bioreactor(MEBR) is illustrated. In this embodiment, the MEBR comprises anenclosed chamber or locker, in fluid communication with a mediareservoir and enclosing a plurality of membrane envelopes. Asillustrated in FIG. 35A, as growth medium enters the MEBR from the mediareservoir, it flows into each of the plurality of membrane envelopes. Asillustrated in FIG. 35B, each membrane envelope comprises a porous innercore material surrounded on each side by a semi-permeable membrane. Asgrowth medium flows through the membrane envelope, it comes into contactwith and flows through the porous inner core material, enabling afilamentous fungal biomat to access the growth medium through thesemi-permeable membrane and thus grow on an outer surface of thesemi-permeable membrane.

It is to be expressly understood that various other configurations ofbioreactors are contemplated and are within the scope of the presentinvention. By way of non-limiting example, one such configuration is a“trough” bioreactor, in which an upward-opening arcuate hydrophilicmembrane having a relatively small pore size (e.g. 0.2 μm) is providedas a trough, and a hose with holes therethrough is providedlongitudinally parallel to the membrane. Feedstock may be provided, e.g.by spraying, through the holes in the hose to ensure that an inner/upperarcuate surface of the trough is evenly saturated with growth medium atall times. Biomass may then grow on the opposite side of the troughmembrane, i.e. an outer/lower surface, and may in embodiments thereby beenabled to fall away from the membrane under its own weight when acertain mass is attained.

Use of the Biomat Reactors in Zero Gravity

The primary physical force controlling formation and growth of thebiomat in the disclosed reactor is attachment to the membrane. Withoutbeing bound by theory, it is believed that biomats grown in thedisclosed reactor will not be impacted by the zero-gravity conditionsexperienced during space flight. Gravity driven directional growth orgrowth controlled by physical mixing or flow is not the overridingfactor in the system, as it tends to be in gravity environments.Previous experiments in space successfully demonstrated fungal growthEuropean Space Agency, Expeditions 25-28, Growth and Survival of ColoredFungi in Space (CFS-A)), providing an additional measure of confidencethat the disclosed reactor system will function in a space environment.

For space missions and ease of deployment, freeze dried inoculum andessential ingredients to support growth on specific feedstocks (ifneeded) can be preloaded in the reactor. Astronauts and space travelerscan then prepare the feedstock, inoculum, and any media components.Incubation time is dependent on the feedstocks, the strain ofmicroorganism, and other growth parameters such as pH, temperature andwater content. The incubation conditions are simple in that fermentationis conducted under static conditions where the reactor is simply allowedto incubate in place. Dense consolidated biomats are harvested by simplyopening the reactor closure (e.g. a ziplock®-type) and removing themats.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims. The examples and figures are provided for the purposeof illustration only and are not intended to limit the scope of thepresent invention. Each publication or other reference disclosed hereinis incorporated herein by reference in its entirety, to the extent thatthere is no inconsistency with the present disclosure.

EXAMPLES Example 1 Growth of Strain Fusarium Strain MK7 and Other Fungiin Static Tray Reactors

Filamentous acidophilic Fusarium strain MK 7, Ganoderma lucidum (Reishi;FIG. 1A), Pleurotus ostreatus (pearl oyster, FIG. 1B: and blue oyster,FIG. 1C), Sparassis crispa (cauliflower; FIG. 1D), Hypsizygus ulmarius(elm oyster; FIG. 1E), Calvatia gigantea (giant puffball; FIG. 1F), andFusarium venenatum biomats were grown in shallow static tray reactors asdescribed in PCT/US2017/020050.

Example 2 Growth of Fusarium Strain MK7 Biomat on Nutrient MediumRefreshed Daily (Semi-Static Conditions)

Dense Fusarium strain MK7 biomats approximately 3 cm thick were grown in21 days on nutrient medium that was refreshed daily. The biomats weregenerated using sterile MK7-1 liquid medium (described inPCT/US2017/020050) containing 7.5% glycerol at pH 3.0 in 12.7×17.8 cmPyrex® glass trays. To initiate the experiment, 200 mLs of the nutrientmedium was inoculated with 5% (volume/volume) of Fusarium strain MK7culture in the late exponential growth phase as described previously inPCT/US2017/020050. 200 mLs of the inoculated medium were added to eachof three sterile trays that were lined with sterile coarse nylon meshscreens. The cultures were incubated undisturbed for 4 days at roomtemperature (˜22° C.) to allow development of the initial biomat layerthat formed at the surface of the liquid. After 4 days of growth, thebiomats were gently lifted out of the tray using the nylon mesh screensand were tilted at a 45 degree angle to allow the liquid to drain out ofthe mats. The biomats were allowed to drain in this position until lessthan one drop of liquid dripped out every five seconds. Sufficientdraining occurred, on average, after about 3 minutes. The drip-driedbiomats in their screens were placed in fresh preweighed 12.7×17.8 cmPyrex® trays containing 200 mL of fresh MK7-glycerol medium (describedin PCT/US2017/020050). Trays with biomats were re-weighed. The processof moving the biomats to another tray containing fresh medium wasrepeated on approximately a daily basis for 17 more days. Sampling ofone of the biomats occurred on days 12, 15 and 21 and the moisturecontents of these biomats were determined. The average moisture contentof the biomats was 17.3% (std dev=0.7) and this value was used tocalculate dry biomass production over the duration of the experiment.Dry biomass production was linear from day 4 through day 18 (r²=0.995)after which biomass weight stabilized at about 2.5 Kg dry/m² (FIG. 1,y-axis normalized to a per m² basis, growth is typically exponentialbetween day 0 and day 4). The average growth rate over this time periodof linear growth was 6.04 g/m²/h. FIG. 2 shows a ˜3 cm thick biomat thatdeveloped after a total of 21 days growth using this method.

To confirm these findings, the same experimental protocol was repeated,this time alongside control trays in which the medium was not refreshedduring biomat growth. The results of this comparative experiment areillustrated in FIG. 36. As FIG. 36 shows, the trays in which the mediumwas refreshed showed biomat growth that was approximately three times asrapid (109 vs. 34 grams dry biomass per square meter per day), for aperiod twice as long (21 vs. 10 days). Surprisingly, it was found thatrefreshing the growth medium did not merely perpetuate the same growthrate as the control, but in fact accelerated growth; where both therefreshed trays and the control showed similar amounts of growth up today 3 (before the medium was refreshed for the first time), a sharpincrease in growth rate was observed in the refreshed trays beginningbetween days 3 and 4, and this increase persisted over nearly the entirelength of the experiment.

Example 3 Growth of Biomats Under Continuous Flow Conditions

A continuous flow bioreactor system was fabricated to demonstrate growthof biomats on the surface of flowing liquid media. The system wasfabricated from a 2.44 m long clear plastic roofing panel with a seriesof corrugations that were used as flow channels (FIG. 3). The ends ofeach of the channels were dammed with silicon (100% Silicone, DAPProducts Inc., Baltimore, Md.) enabling liquid to be retained within thechannels. Flow was facilitated through the channels by delivery ofliquid media to one end of the channels via a peristaltic pump, with theliquid exiting the other end of the channels through holes in the bottomof the channels. The whole plastic roofing panel system was slanted atan angle of 1 cm rise per 1 m run to enable about 500 mL of liquid to beretained in each channel and a consistent flow being a function of theamount of liquid and the angle of the inclination.

The panel system was sanitized and wrapped in Saran®-like plastic wrapto isolate the system from the surrounding room environment. Sterile airwas pumped under the plastic wrap at a rate of 400 mL/min creating apositive pressure on the system. To initiate development of a biomatprior to starting flow, a 500 mL volume of nutrient medium inoculatedwith the desired filamentous fungus was added per channel and allowed toincubate under quiescent/static conditions for 4 days. After 4 days, theperistaltic pump delivered a continuous pulsed flow of 400 mL/d to“feed” the biomats (ON at 2.016 mL/min for 49 min, 39 sec; OFF for 5 h10 min 21 sec). Two independent experiments were conducted with eachexperiment using two separate flow channels as replicates (FIG. 3).

Consolidated biomats were harvested after 10 days of growth on thenutrient medium (4 days under quiescent/static conditions followed by 6days under continuous flow; FIG. 4). Average dry weight of the producedbiomass was an average of 2.38 g for the replicate flow channels. Duringthe continuous flow periods (day 4 to day 10) the average removal ratesof C and N from the flowing liquid medium by the growing biomats were11.9 and 1.2 mg/L/h, respectively. C and N removal rates from the liquidmedium were determined by measuring liquid volume and total C and Ninputs and outputs from the bioreactor system using a Costech total Cand N analyzer (ECS 4010, Costech Analytical Technologies, Valencia,Calif.). Thus, the continuous flow system supported biomat growth at thesurface. The experiments also served as a laboratory-scale demonstrationfor continuous feed of Fusarium strain MK7 biomat growth and productionof consolidated biomats. It should be noted that other feedstocks, flowrates and resulting growth rates can be achieved with this type ofsystem. For example, with 10% glycerol in MK7-1 medium (described inPCT/US2017/020050) at pH 2.8, expected yields are greater than 40 gramsdry biomass per day per m².

Example 4 Semi-Continuous and Continuous Production of Fusarium StrainMK7 Biomats

Dense Fusarium strain MK7 biomats were grown and harvested on asemi-continuous basis over a period of 19 days. The biomats weregenerated using acid whey as the feedstock/carbon source supplementedwith ½ strength MK7-1 medium salts (described in PCT/US2017/020050)adjusted to pH 4.0. To initiate the experiment, 200 mL of the nutrientmedium inoculated with Fusarium strain MK7 (5% volume/volume) in thelate exponential growth phase was added to sterilized 12.7×17.8 cmPyrex® glass trays, which were then covered with Saran® wrap andincubated at room temperature. After 5 days of growth, ⅓ of the biomatfrom one end of the tray was removed by cutting and removing a 5.9×12.7cm section of biomat (FIG. 5A). The remaining ⅔ of biomat was thenphysically moved over to the open area of medium that was created byremoval of the ⅓ portion of biomat. The biomat was shifted by physicallygrasping it with sterile gloved fingers and pulling the biomat overuntil it touched the end of the tray to open medium with no formedbiomat at the other end of the tray (FIG. 5B). The process of harvestinga ⅓ section of the most mature portion of the biomat and then moving theremaining ⅔ of biomat over the open area was repeated periodically. 50mLs of medium were aseptically removed from the tray every 4 days andreplaced with 50 mLs of fresh sterile medium (acid whey with ½ strengthMK7-1) to replenish the nutrients removed from the liquid medium byremoval of the biomat. Dry biomass production using this method yielded0.57 g/day per tray or 25.2 g/d/m² between days 5 and 19 (FIG. 6). Thus,a semi-continuous production system was demonstrated whereby the mostmature end of the biomat was harvested at an average rate of 1.56 cm/dayand fresh biomat growth was initiated in the open area of medium at theother end of the tray.

The system is also amenable to continuous harvesting and growth of abiomat whereby continuous removal is facilitated by a roller wheel thatis attached to the mature end of the biomat (FIG. 7). The roller wheelslowly turns and harvests the mature biomat and at the same time createsan open medium for growth of new biomat at the other end of the tray.The roller wheel turns and harvests the biomat at a rate of 1.56 cm/dayto reproduce the semi-continuous system described above. It is desirablethat the nutrients in the liquid medium be replenished at the rate ofnutrient removal by the biomat.

Example 5 Membrane Encapsulated Bioreactors

Dense Fusarium strain MK7 biomats were grown in liquid growth mediumthat was encapsulated in a bioreactor system with no gas headspace.Sterile Petri dish bottoms (55 mm diameter) were filled to the brim with57 mL of inoculated MK7-1 medium (described in PCT/US2017/020050)containing 8% glycerol. Gas permeable/semi-permeable membranes ofpolypropylene and polycarbonate were placed directly on the surface ofthe liquid medium and sealed tightly with rubber bands. No gas headspacewas provided at the start of the growth period.

After inoculating the medium and sealing the membranes, the bioreactorswere allowed to sit undisturbed until harvest. FIG. 8 shows the ˜5 mmand ˜1 mm thick biomats of Fusarium strain MK7 that grew directlyunderneath the polypropylene (FIGS. 13A-13C) and polycarbonate (FIG.13D) membranes in five days, respectively. The biomats mildly adhered tothe membranes and could be easily harvested by simply peeling away thebiomats from the membranes (FIG. 13).

Example 6 Production of Pigments and Vitamin D2 by Irradiation ofFusarium MK7 Biomats with UVB

UVB light (290-320 nm) was used to trigger pigment production byFusarium strain MK7 biomats. Fusarium strain MK7 biomats produced in 3days on 7.5% glycerol MK7-1 medium (described in PCT/US2017/020050) wereirradiated with UVB light for a period of 4 hours. The UVB light wasemitted from a 50 W bulb (Slimline Desert 50 UVB T8 fluorescent bulb, 46cm; Zilla, Franklin, Wis.) placed 10 cm above the biomat. Orangepigmentation was visually detected after 0.5 h of irradiation and waspronounced after 4 h of irradiation (FIG. 9). In addition, biomats thathave not been exposed to UVB light have a vitamin D2 content of lessthan 50 IU/100 g of biomat whereas after UVB light exposure forapproximately 12 hours the vitamin D2 content is increased toapproximately 1.2 million IU/100 g biomat.

Example 7 Fusarium Strain MK7 Biomats Grown on a Mixture of Glycerol,Starch and Corn Steep Liquor

Fusarium strain MK7 biomats were produced from a mixture of glycerol,starch, corn steep liquor and MK7-1 salts (described inPCT/US2017/020050) in as little as 4 days. Glycerol was purchased fromDuda Energy LLC (Decatur, Ala.; 99.7% Purity; USP Grade; Lot#466135376340); 100% Argo Corn Starch manufactured by Argo FoodCompanies, Inc (Memphis, Tenn.) was purchased from Albertson'ssupermarket in Bozeman, Mont., and the corn steep liquor was purchasedfrom Santa Cruz Biotechnology, Inc. (Dallas, Tex.; Lot #B0116). Thegrowth medium was a mixture of 7.5% glycerol (weight/weight), 2.5%starch and 2.5% corn steep liquor with MK7-1 salts. The mixture wasadjusted to pH 3.3 by adding an appropriate amount of HCl and boiled for15 minutes in a suitable container. After cooling to room temperature,the pH of the mixture was readjusted to 3.3 and then inoculated with 5%Fusarium strain MK7 inoculum (vol/vol) as prepared in PCT/US2017/020050.Aliquots of 1.5 L inoculated media were added to three sanitized 0.25 m²polypropylene trays, placed in a sanitized tray rack system that wascompletely covered with aluminum foil to create dark conditions, andincubated at 23°±1° C. The filamentous fungal biomats that grew at thesurface of the medium were harvested after 4 days by simply lifting thebiomats from the trays.

The average final pH of the residual liquid in the three trays was 4.45(standard deviation=0.14). Three 56.7 cm² circular portions were cut outand removed from each of the biomats at random positions and theseportions were dried at 50° C. for 48 h to obtain dry weights. Theaverage biomass dry weight (standard deviation) was 124.6 g/0.25 m²(43.4) or 498.4 g/m² (173.6). The mean thickness of the moist biomatswere 7.5 mm and the mean density on a dry weight basis was 0.66 g/cm³.

To expose the biomat filaments and enable examination by Field emissionscanning electron microscopy (FE-SEM), the extracellular matrix (ECM)between the filaments were removed by washing with ethanol. Toaccomplish this, 1 cm² portions (1 cm×1 cm) of the biomats were excisedwith a razor blade immediately before harvesting, and the excisedportions were subjected to an ethanol washing/dehydration series bysequentially submersing the samples for the noted times in 40 mL of theethanol mixtures as follows: 25% ethanol, 75% deionized H₂O for 20minutes; 50% ethanol, 50% deionized H₂O for 20 minutes; 75% ethanol, 25%deionized H₂O for 20 minutes; 95% ethanol, 5% deionized H₂O for 20minutes; 100% ethanol, 0% deionized H₂O for 60 minutes. The 100% ethanoltreatment was repeated 2 more times before storing the samples in 100%ethanol.

To retain microstructure integrity of the biomats for FE-SEM, ethanolwashing/dehydration was followed by critical point drying using aTousimis Samdri-795 critical point dryer according to the manufacturerinstructions (Tousimis Samdri-795 Operations Manual; Tousimis,Rockville, Md.). After critical point drying, the samples were eithermounted directly onto aluminum stubs or sliced into <0.3 mm thicksections with a razor blade prior to mounting. The samples were thencoated with iridium (20 μm, EMITECH K575X, Electron Microscopy Sciences,Hatfield, Pa.) and examined with a JEOL 6100 FE-SEM using an incidentbeam energy of 1 keV (JEOL USA, Inc., Peabody, Mass.).

FE-SEM imaging revealed a complex network of interwoven hyphal filaments(FIG. 10), very similar to the structure revealed by light microscopyfor biomats grown on glycerol as reported in PCT/US2017/020050. Threedistinct layers were observed: (a) an aerial hyphae layer at the topsurface, (b) a dense bottom layer and (c) a transitional layer betweenthe top and bottom layers. The transitional layer was only looselyattached to the dense bottom layer, thus enabling easy separation of thebottom layer from the rest of the biomat. Filament densities of thetransitional layer ranged from slightly less dense than the bottom layerin the zone where the two layers met, to a density that was comparableto the aerial hyphae near the top of the biomat.

Excised samples were also prepared for light microscopy by slowlydipping into the following solutions in the order and times shown below:

Xylene, 3 min; Xylene, 3 min; 100% ethanol, 3 min; 100% ethanol, 3 min;95% ethanol, 3 min; 95% ethanol, 3 min; 70% ethanol, 3 min; Deionizedwater, 3 min; Hematoxylin 1, 1.5 min; Running tap water rinse, 1 min;Clarifier solution, 1 min; Running tap water rinse, 1 min; Bluingsolution, 1 min; Running tap water rinse, 1 min; 70% ethanol, 30 dips;95% ethanol, 30 dips; 95% ethanol, 30 dips; 100% ethanol, 30 dips; 100%ethanol, 30 dips; 100% ethanol, 30 dips; Xylene, 30 dips; Xylene, 30dips; Xylene, 30 dips; Apply cover slip.

Sections of the biomats approximately 2 cm² in size were excised fromthe fresh biomats with a razor blade immediately before harvesting.These sections and then immersed in 35 mL of deionized water in 50 mLconical bottom centrifuge tubes. The tubes were sonicated (CP200TUltrasonic Cleaner, Crest Ultrasonics, Ewing, N.J.) for either 0, 40, 90or 150 seconds to disperse filaments into the liquid and enablemicroscopic observation. Aliquots of the liquid (˜100 uL) from thesetubes were placed on a glass slide, covered with a cover slip andobserved with a light microscope (B400B, Amscope, Irvine, Calif.) at100× magnification. The average length (std dev) of non-broken filamentswere measured and determined to be 1.1 (0.6), 1.2 (0.4), 1.0 (0.4) and1.2 (0.2) mm for the 0, 40, 90 and 160 second sonication treatments,respectively. The maximum filament length observed in each treatmentwere 2.5, 1.4, 1.8, and 1.4 mm, respectively. These filament lengths aresignificantly longer compared to growth of Fusarium strain MK7 insubmerged shake flask cultures where average lengths are less than 0.02mm.

Example 8 Production of Chicken Nuggets Using Fusarium Strain MK7Biomats Grown on a Mixture of Glycerol, Starch and Corn Steep Liquor

Fusarium strain MK7 biomat, produced as described above, were used tocreate chicken nuggets. Moist biomats were steamed in a pot steamer at97° C. for 0.5 hour, cooled to room temperature and used as the base toproduce chicken nuggets. Steamed moist biomat (200 g) was chopped intopieces less than 0.5 mm long and homogenized with 4% (weight/weight; 8g) chicken base and 4% egg white protein (8 g). The resulting mixturecomprised more than 90% Fusarium strain MK7 biomat. Portions of thisbiomat mixture (˜30 g) were formed into nugget shapes and steamed for ina pot steamer. The prepared nuggets were breaded by coating in eggwhites and then mixing with bread crumbs that adhered to the surfaceprior to frying. The prepared nugget exhibited a chicken meat liketexture and exuded the typical aroma of chicken. Taste testing by 5people deemed the nugget to closely simulate actual chicken containingchicken nuggets in terms of taste and texture.

Example 9 Production of Fusarium Strain MK7 Biomat Extract

Highly concentrated and viscous extracts were produced from Fusariumstrain MK7 biomats. Biomats harvested after 4-16 days of cultivation, aspreviously described, are rinsed and steamed, drip dried on porousplastic mesh for 5 minutes, and placed in plastic bags and sealed.Sealed bags are frozen at either −20° C. or −80° C. for 24 hours priorto being incubated at 60° C. incubator in the original sealed bags for48 hours after pH adjustment of the remaining medium liquid to betweenpH 4-6. After heat treatment, biomats are pressed through <1.5 mm poresize filters and the resulting liquid collected. The collected liquid isboiled for 10 minutes in a non-reactive vessel then dried at 60° C.until water content is ˜6-8%, forming a sticky paste extract. Thenutritional value of the extract is similar to the nutritional value ofthe steamed biomat and flour made from steamed biomats.

Example 10 Production of Yogurt From Fusarium Strain MK7 Biomats Grownon Acid Whey

Fusarium strain MK7 biomats were used directly to produce yogurt. Thebiomats were grown in trays on an acid whey feedstock/carbon source thatwas generated as a by-product of Greek yogurt manufacture, harvestedafter 6 days and were steamed within 20 minutes of harvesting. 200 g ofthe cooled, moist biomass was blended together with 600 g of drinkingquality tap water to produce a milk-like suspension referred to as “MK7liquid dispersion.” The MK7 liquid dispersion was used as an ingredientby itself or in combination with cow's milk to produce yogurt.

Three mixtures containing different ratios of MK7 liquid dispersion towhole milk were prepared: 1) 25% MK7 liquid dispersion:75% whole milk,2) 50% MK7 liquid dispersion:50% whole milk, and 3) 100% MK7 liquiddispersion. The mixtures were used to make three batches of yogurt byheating each mixture to 83° C. and holding at that temperature for 14minutes with constant stirring. The mixtures were allowed to cool to 43°C. and then live yogurt cultures added as inoculum. The resultingmixture was incubated at 44° C. in a yogurt maker (Model YM80;EuroCuisine, Los Angeles, Calif.) for 8 hours. All of the resultantmixtures had the appearance and texture of yogurt, as well as a smelland taste similar to typical yogurt.

Example 11 Growth of Mushroom Biomats on Glycerol

Biomass biomats comprised of Baby Bella Brown Crimini Mushrooms(Agaricus bisporus) and White Mushrooms were produced in as little as 10days using glycerol as the primary carbon source (feedstock). Thesecommon edible mushrooms were purchased from Albertson's supermarket inBozeman, Mont. and stored at 4° C. The medium used to grow the mushroomsconsisted of 1 L of 7.5% glycerol with MK7-1 salts (described inPCT/US2017/020050) that was boiled for 10 minutes followed by cooling toroom temperature (˜23° C.). The pH of the mixture was adjusted to 2.7and 200 mL of the pH adjusted mixture was poured in two sterile12.7×17.8 cm Pyrex® trays. The inoculum consisted of 5 g of blended,surface-sterilized Crimini or White Mushrooms that was added to themedium in each tray. The mushroom inoculum was prepared as follows: 1)10 g of moist Crimini or White Mushrooms were added to 200 mL of a 5%bleach solution and the suspension was stirred for 2 minutes to surfacesterilize the mushrooms, 2) the mushrooms were then rinsed bytransferring into 200 mL of sterile glycerol/MK7-1 salts medium(described in PCT/US2017/020050) and stirring for 2 minutes, 3) thesurface sterilized mushrooms were blended for 30 seconds in a coffeegrinder that had been sterilized by rinsing with 70% ethanol, 4) theground mushroom biomass (<5 mm long aggregates) was surface sterilizedagain by repeating steps 1 and 2 with the ground biomass, 5) 5 grams ofthe ground mushroom biomass was added to the liquid medium in the Pyrex®trays (final pH=4.0-−4.1 after addition of mushrooms), and 6) the trayswere covered and allowed to incubate at room temperature (22±2° C.) inthe dark.

Biomats were observed to develop on the surface of the medium after 3days of incubation and consolidated biomats were harvested after 10 daysof growth. Biomats of Crimini Mushrooms covered the entire surface ofthe liquid medium in the tray while biomat growth of White Mushroomscovered approximately ½ the liquid medium as five floating biomatislands. The mean thickness of the biomats were 1.5 mm for the Criminiand 1.7 mm for the White Mushrooms. Biomass biomats were dried at 50° C.for 48 h and the dry weights produced per tray were 1.14 g and 2.12 gfor the Crimini and White Mushrooms, respectively. Densities on a dryweight basis for the dry biomass biomats were 0.033 and 0.111 g/cm³ forthe Crimini and White Mushrooms, respectively.

Microscope images revealed the mycelial nature of the biomats. Averagehyphal thicknesses were 25.2 μm (std dev=6.2) and 18.7 μm (4.0) for theCrimini and White Mushroom biomats, respectively.

Produced Crimini biomats were used to create chicken nuggets. Biomatswere steamed at 97° C. for 0.5 hour, cooled to room temperature and usedas the base to produce chicken nuggets. Steamed moist biomass (2.5 g)was mixed with 3% (weight/weight; 75 mg) Better Than Bouillon chickenbase (Southeastern Mills, Inc. Rome, Ga.) and 3% Eggwhite Protein (75mg; Now Foods, Bloomingdale, Ill.) and chopped into pieces less than 2mm long using a razor blade. The mixture was formed into a nugget andsteamed for 0.5 hour. The prepared nugget provided the typical aroma ofchicken with a slight mushroom fragrance. When tasted, the nugget had achicken to neutral flavor.

Example 12 Growth of Mushroom Biomats on Malt and Glycerol Media

Biomass biomats comprised of Calvatia gigantea (giant puffball),Pleurotus ostreatus (pearl oyster), Pleurotus ostreatus var. columbinus(blue oyster), Hypsizygus ulmarius (elm oyster), Sparassis crispa(cauliflower) and Ganoderma lucidum (reishi) were produced in as littleas 5 days using Malt Extract Medium 001, Glycerol Medium 002, Hansen'sMedium, MK7-SF Medium, Malt Extract+NH₄NO₃ Medium 003 (Table 3). Allfinal media contained 0.01% chloramphenicol.

TABLE 3 Ingredients added to deionized or drinking quality tap water toprepare nutrient media. Malt Extract Medium 001 Ingredient Amount GradeLot # Vendor Location Light Pilsner 40.0 g Food 180526BHomebrewstuff.com Boise, ID Malt Peptone 4.0 g Research 44984-57374Research Products Mt. Prospect, International IL Yeast Extract 1.2 gResearch 53852-66581 Research Products Mt. Prospect, PowderInternational IL Canola Oil 1.0 mL Food SEP/25/19 Better Living LLCPleasanton, CA S3283 CA Ground Oats 4.0 g Food Jan 25, 2020Walmart-Stores, Inc Bentonville, I2M 06:36 AR Tap H₂O 1000 mL N/A N/AN/a Bozeman, MT Glycerol Medium 002 Ingredient Amount Grade Lot # VendorLocation Glycerol 40.0 g Food/USP 20149018137001 Duda Energy Decatur,LLC AL Peptone 4.0 g Reagent 44984-57374 Research Mt. Prospect, ProductsIL International Yeast 1.2 g Reagent 53852-66581 Research Mt. Prospect,Extract Products IL Powder International Canola Oil 1.0 mL FoodSEP/25/19 Better Living Pleasanton, CA S3283 LLC CA Ground 4.0 g FoodJan 25 2020 Walmart- Bentonville, Oats I2M 06:36 Stores, Inc AR Tap H₂O1000 mL N/A N/A N/a Bozeman, MT Hansen's Medium Ingredient Amount GradeLot # Vendor Location Peptone 1.0 g Reagent 44984-57374 Research Mt.Prospect, Products IL International KH₂PO₄ * 7H₂O 0.3 g Reagent Mfg.Doesn't Eisen-Golden Dublin, use lot numbers Laboratories CA MgSO₄ *7H₂O 2.0 g USP 81721 San Francisco San Leandro, Salt Co. CA Glucose 5.0g Reagent 0435C235 Fisher Denver, Scientific CO Tap H₂O 1000 mL N/A N/AN/a Bozeman, MT MK7-SF Medium Ingredient Amount Grade Lot# VendorLocation NH₄NO₃ 7.553 g ACS A0390194 Acros Organics Somerville, NJ Urea2.548 g USP 30570-67229 Research Mt. Prospect, Products IL InternationalCaCl₂ 2.000 g Reagent 102615 Fritz Pro Mesquite, Aquatics TX MgSO₄ *7H₂O 2.000 g USP 81721 San Francisco San Leandro, Salt Co. CA KH2PO47.500 g Reagent Mfg. Doesn't Eisen-Golden Dublin, use lot numbersLaboratories CA Trace * 2.000 mL * * * * Glycerol 0.075 Kg Food/USP20149018137001 Duda Energy Decatur, LLC AL Yeast 1.750 g Research53852-66581 Research Mt. Prospect, Extract Products IL InternationalFeCL₂ * 4H₂O 0.020 g Reagent 951164 Fisher Fair Lawn, Scientific NJ DIH₂O 0.940 L N/A N/A N/A Bozeman, MT Trace Components * Micronutrients*mg/L Grade Lot # Vendor Location FeSO4•7 H2O 9.98 ACS 3562C398 AmrescoSolon, OH ZnSO4•7 H2O 4.4 USP/FCC 61641 Fisher Waltham, MA MnCl2•4 H2O1.01 Reagent 13446-34-9 Fisher Waltham, MA CoCl2•6 H2O 0.32 Reagent7791-13-1 Fisher Waltham, MA CuSO4•5 H2O 0.31 Technical 114675 FisherWaltham, MA (NH4)6Mo7O24•4 H2O 0.22 ACS 68H0004 Sigma St. Louis, MOH3BO3 0.23 ACS 103289 Fisher Waltham, MA EDTA, free acid 78.52Electrophoresis 46187 Fisher Waltham, MA Malt Extract + NH₄NO₃ Medium003 Ingredient Amount Grade Lot # Vendor Location NH₄NO₃ 5.0 g ACSA0390194 Acros Organics Somerville, NJ Light Pilsner 40.0 g Food 180526BHomebrewstuff, com Boise, ID Malt Peptone 4.0 g Research 44984-57374Research Products Mt. Prospect, International IL Yeast Extract 1.2 gResearch 53852-66581 Research Products Mt. Prospect, PowderInternational IL Canola Oil 1.0 mL Food SEP/25/19 Better Living LLCPleasanton, CA S3283 CA Ground Oats 4.0 g Food Jan 25, 2020Walmart-Stores, Inc Bentonville, I2M 06:36 AR Tap H₂O 1000 mL N/A N/AN/A Bozeman, MT

The above recipes in Table 3 were used to prepare media in either 2 LPyrex® bottles or 8 L stainless steel pots by mixing the specifiedingredients into the specific volumes of water depending on the volumeof media desired. Ingredients were added to water while liquid wascontinuously stirred with a stir bar or a spoon. Each component of themedia was thoroughly mixed into the liquid before the next component wasadded, pH for the MK7-SF medium was adjusted to 5.0, and the solutionsautoclaved. All other pH's resulted from simply mixing the ingredients.The medium and vessels were autoclaved for at least 20 minutes at 20 psiand 121° C. Osmotic pressure (as osmolality) of the liquid was measuredusing an Advanced Instruments, Inc. osmometer Model 3250 (Two TechnologyWay, Norwood, Mass.).

After autoclaving, the media were allowed to cool to room to temperatureand individual vessels were inoculated with the mushroom species shownin Table 4.

TABLE 4 Mushroom spores (10 cc syringes) were purchased from MycoDirect(12172 Route 47, Ste 199 Huntley, Il 60142) and received on Aug. 2,2018. Elm Oyster spores were purchased from Everything Mushrooms (1004Sevier Ave Knoxville, TN 37920) and received on Aug. 3, 2018. Lot DateProduced by Company Blue Oyster 3-P7 February-2018 Pearl Oyster 9P8December-2017 Giant Puffball N/A March-2018 Cauliflower Mushroom N/AApril-2018 Elm Oyster (1 cc dried) N/A October-2017

Inoculation of growth media was preformed using the following methodsapplied using aseptic technique. All aseptic work in these experimentswere performed in Class II biosafety cabinet. Spore syringes were usedto directly inoculate approximately 75 mL of growth medium in previouslyautoclaved, 12.7×17.8 cm Pyrex® glass trays. This was done byaseptically transferring liquid medium into an autoclaved Pyrex® tray,allowing the media to cool to room temperature and inoculating with 2 ccof the suspension contained in the spore syringe. The tray was coveredwith sterile aluminum foil and then gently swirled to mix the inoculatedmedium.

Malt Extract Agar (MEA; Table 5) plates were prepared aseptically byautoclaving MEA, allowing to cool to 50° C., and pouring ˜25 mL into100×15 mm sterile Petri dishes.

TABLE 5 Ingredients used to prepare Malt Extract Agar Malt Extract Media(MEA) Ingredient Amount Grade Lot # Vendor Location Light Pilsner 30.0 gFood 180526B Homebrewstuff.com Boise, ID Malt Agar 20.0 gMicrobiological 2170501 BD Sparks, MD Tap H₂O 1000 mL N/A N/A N/ABozeman, MT

MEA plates were inoculated by aliquoting 1 cc of liquid from thesuspension contained within the spore syringe onto the plates. The agarplates were then sealed with Parafilm® and placed into a clean darkdrawer at room temperature.

After mycelium had covered the entire surface of the MEA plates, theywere used for inoculation of 1.5 L medium in 2 L baffled shaker flasks.Approximately 2 cm² portions of agar medium with mycelium on the surfacewere excised from the plates with a sterile razor blade and diced into˜2 mm² portions, which were then added to two flasks containing 1.5 L ofMalt Extract 001 medium. The medium was incubated for 3 days at roomtemperature (23±1° C.) with intermittent shaking by hand (flasks werevigorously shaken by hand for 1 minute at a minimum of five times perday).

The cultures in the shaker flasks were then used as inoculum for 6 L ofMalt Extract medium 001 and for 6 L of Malt Extract+NH₄NO₃ 003 medium.The media were inoculated with 15% (vol:vol) of inoculum culture andmixed thoroughly. Two liters of inoculated media were poured into eachof three 0.25 m² plastic trays that were placed into a tray rack. Theracks were wrapped in Saran® and allowed to incubate for 6 days.Relatively dense biomats covering the entire surface within 4 days andthe biomats were harvested after 6 days.

Biomats from 12.7×17.8 cm Pyrex® glass trays and the 0.25 m² plastictrays were harvested by lifting the biomats from the trays and gentlysqueezing by hand. Portions of the biomats (3-50 g) were streamed for 20minutes over boiling water (˜5 cm above surface of water) in a potsteamer set on a kitchen oven burner. After steaming, the biomass wasallowed to cool to room temperature and immediately bagged in a Ziploc®bag and sent to Eurofins (Des Moines, Iowa) for protein analysis (N bycombustion, Test Code QD252).

TABLE 6 Results from a series of different filamentous fungi growth intrays in various types of media Biomass Wet Ionic Osmotic Final perBiomat Tray Initial Strength Pressure pH Surface Tensile Size Medium(mmol/ (mOsm/ Time Free Area Density Strength Media (m²) pH C:N L) kg)(days) Liquid (g/m²) (g/cm³) (g/cm³) Giant Puffball Malt 001 0.022 6.2819 33.1 169 5.7 5.62 71.4 0.057 314.1 Glycerol 002 0.022 6.96 30 13.6505 5.7 5.54 40 0.04 214.9 Hansen's 0.022 8.81 27 30.7 39 n/a n/a n/an/a n/a MK7-SF 0.022 4.91 7.5 344 1387 9.0 5.07 178.6 0.045 135.0 Malt001 0.25 6.96 19 33.1 169 6.2 6.25 111.1 0.037 264.0 Malt + NH₄NO₃ 0.256.88 7.5 145.1 287 5.8 n/a 108.3 0.11 281.1 003 Cauliflower Malt 0010.022 6.28 19 33.1 169 6.7 4.44 146.7 0.073 507.83 Glycerol 002 0.0226.96 30 13.6 505 6.7 5.77 24.2 0.012 242.91 Hansen's 0.022 8.81 27 30.739 N/A N/A N/A N/A N/A MK7-SF 0.022 4.91 7.5 344 1387 N/A N/A N/A N/AN/A Malt 001 0.25 6.96 19 33.1 169 N/A N/A N/A N/A N/A Malt + NH₄NO₃0.24 6.88 7.5 145.1 287 N/A N/A N/A N/A N/A Blue Malt 001 0.022 6.28 1933.1 169 10 5.7 112.5 0.023 72.34 Glycerol 002 0.022 6.96 30 13.6 505 105.56 56.1 0.014 37.37 Hansen's 0.022 8.81 27 30.7 39 N/A N/A N/A N/A N/AMK7-SF 0.022 4.91 7.5 344 1387 N/A N/A N/A N/A N/A Malt 001 0.25 6.96 1933.1 169 N/A N/A N/A N/A N/A Malt + NH₄NO₃ 0.24 6.88 7.5 145.1 287 5.8N/A N/A N/A N/A Pearl Malt 001 0.022 6.28 19 33.1 169 10 5.47 124.40.025 98.97 Glycerol 002 0.022 6.96 30 13.6 505 N/A N/A N/A N/A N/AHansen's 0.022 8.81 27 30.7 39 N/A N/A N/A N/A N/A MK7-SF 0.022 4.91 7.5344 1387 N/A N/A N/A N/A N/A Malt 001 0.25 6.96 19 33.1 169 N/A N/A N/AN/A N/A Malt + NH₄NO₃ 0.24 6.88 7.5 145.1 287 5.8 N/A N/A N/A N/A ElmMalt 001 0.022 6.28 19 33.1 169 10 5.21 111.6 0.032 143.67 Glycerol 0020.022 6.96 30 13.6 505 N/A N/A N/A N/A N/A Hansen's 0.022 8.81 27 30.739 N/A N/A N/A N/A N/A MK7-SF 0.022 4.91 7.5 344 1387 N/A N/A N/A N/AN/A Reishi Malt 001 0.022 6.28 19 33.1 169 6.7 4.59 0.006 0.13 101.05Glycerol 002 0.022 6.96 30 13.6 505 6.7 4.54 N/A N/A N/A Hansen's 0.0228.81 27 30.7 39 N/A N/A N/A N/A N/A MK7-SF 0.022 4.91 7.5 344 1387 N/AN/A N/A N/A N/A

Example 13 Fusarium Strain MK7 Chicken Nugget

Chicken flavored Fusarium strain MK7 is a basic ingredient to a numberof recipes including chicken nuggets, with or without breading, chickenfor Asian dishes, or other chicken dishes as a chicken replacement.Fusarium strain MK7 biomats produced from different feedstocks/carbonsources result in slightly different chicken flavors. The glycerolchicken is sweeter and the acid whey chicken tends to be a little bitsourer.

The amount of food processing and the blade used (i.e. sharp metalblade, dull metal blade, plastic blade) result in different chickennugget textures. Further, acceptable chicken nuggets can be producedfrom a wide variety of biomass sizes. That is, biomass can be cut with aknife, lightly food processed or highly food processed and still resultin acceptable chicken analogs.

A 50-20:1:1 ratio of Fusarium strain MK7:chicken stock:binder was usedwith or without approximately a 66.6% Fusarium strain MK7:fat ratio.Suitable fats include duck fat, coconut butter, and cocoa butter. Aftermixing, the mixture is steamed for approximately 30 minutes to set thebinder; however, some binders may require more or less time. Additionalbreading can then be added and the resulting nuggets process as typicalfor such foodstuffs.

Example 14 Breakfast Sausage and/or Hot Dog and/or Burger

An appropriate spice mix is added to size reduced Fusarium strain MK7biomats as needed to develop the flavors desired, which may be between10 wt. % of spice mix to a quantity of Fusarium strain MK7 up to 20%,oftentimes in a ratio of 10 Fusarium strain MK7:1 spice mix, with orwithout additional ingredients such as onion, binders, and a fat such ascocoa butter. The mixture is then fried to remove an appropriate amountof moisture. Additional ingredients can then be added, such as bulgur,vegetable broth, potatoes, etc. prior to shaping in the desired shapeand cooking.

Example 15 Ice Cream and Mousse

A ratio of approximately 1:3 Fusarium strain MK7 biomat:water isgenerated having a particle size with average filament lengths less than900 microns. This mixture is gently heated until there is no longer afungal scent and then used in approximately a 4:1 ratio with cashews,optionally with an appropriate amount of xanthan gum and/or flavoring,to generate a mix which may be optionally heated and then cooled to forma mousse. For frozen dessert, the mix is then placed in an ice creamchurner and, after churning, frozen to form a non-meltable frozendessert.

Example 16 Production of Truffle Oil from Truffle Biomats

Oil extract can be prepared from Truffle (Tuber sp.) biomats grown asdescribed above. In one instance, truffle biomats were grown in trays inas little as 7 days using malt extract, glucose and peptone as theprimary carbon sources (feedstock). The edible Truffle mushroom waspurchased from IGB Trading LLC on the Amazon Marketplace and stored at4° C. A pure culture of the Tuber sp. fungus was prepared from thepurchased truffle by placing ˜3 mm³ portions of truffle (cut with asterile razor blade) on Malt Extract Agar+0.01% chloramphenicol (used toinhibit bacterial growth). A Malt Extract Agar was prepared by mixing 20g of malt extract, 20 g of glucose, 1 g peptone and 20 g of agar in 1 Lof deionized water prior to autoclaving for 30 minutes and cooling to50° C. before adding 0.01% chloramphenicol. The sterile mixture was thenpoured into 9 cm diameter Petri plates and allowed to cool and solidify.

The fungus was observed to grow on the trays after 3 days. After 4 daysof growth, hyphae were picked with a sterile microbiological loop andstreaked onto a fresh set of Malt Extract Agar+chloramphenicol plates.The fungus was allowed to grow on said plates for 5 days, after whichhyphae were picked with a microbiological loop and used to confirmculture purity by DNA sequencing. Confirmation was accomplished byextracting and purifying the DNA (FastDNA Spin Kit, MP Biomedicals) andsequencing the ITS region of the metagenome followed by phylogeneticclassification of the sequences using Blast (NCBI database).

Malt Extract Broth was prepared by mixing 20 g of malt extract, 20 g ofglucose and 1 g peptone in 1 L of deionized water and sterilized.Scrapes of the hyphae with the microbiological loop were also used toinoculate 50 mL of sterile Malt Extract Broth in sterile baffled shakerflasks capped with sterile gauze material. Sterile gauze was used as itallowed exchange of gases into and out of the shaker flask. Shakerflasks were then rotated at 185 rpm for 5 days. The rotated cultureswere then used to inoculate 350 mL of sterile Malt Extract Broth insterile 12.7×17.8 cm Pyrex® glass trays. The inoculum density was forthis culture medium was 7.5% inoculum to 92.5% broth. After 7 days ofgrowth in the trays, the filamentous biomat formed on the surface washarvested by lifting the biomat from the liquid medium. The harvestedbiomats were dried at 40° C. for 48 h. Lipids/oil from these harvestedbiomats were extracted by either mechanical pressing or by solventextraction using hexane, although other extraction methodologies can beused.

Example 17 MK7 Flour

Fusarium strain MK7 biomat, produced as described above, was used tocreate dried powder similar in particle size and particle sizedistribution to a standard baking flour. Here, moist biomats weresteamed in a pot steamer at 97° C. for 0.5 hour, cooled to roomtemperature and dehydrated in a Cuisinart dehydrator (model DHR-20) for2-8 hours with an average dehydration time being 4 hours. Dehydrationtime is a function of the amount of biomass loaded into the dehydrator,distribution of biomats in the dehydrator which impacts air flow in thedehydrator and the water content of biomats (average water contentapproximately 75%) and room temperature. Water content post dehydrationvaries between 4 and 14% with average water content post dehydrationbeing below 12%. Dehydrated biomass was size reduced using a coffeegrinder (KRUPS, Electric coffee and spice grinder, stainless steelblades F2034251) until finely ground. Average particle size for groundbiomat flour ranged from 75 microns to 120 microns. A small fraction oflarger particles, app 5 wt %, had a particle size of greater than 180microns. A small fraction of smaller particles, app. 5 wt % had aparticle size smaller than 75 microns. Said smaller particles were of asize which enabled the small particles to remain air borne for extendedperiods of time. Particle size was determined by sifting 100 gramsamples of size reduced biomats for 5 minutes in sieves with 180 μm, 120μm and 75 μm openings. Water content post dehydration and post sizereduction below 6% is preferred as higher water contents can lead toclumping of dried and milled biomass.

Biomat flour was then used as an addition to other standard flours (KingArthur flour, Bob's Red Mill Flour & Bob's Red Mill Wheat Flour) and avariety of baked goods where prepared. Biomat flour was loaded at 5wt %,10 wt %, 20 wt % and 30 wt % with no deleterious effect on ultimatebaked good taste, rising, texture, appearance or smell. Productsdemonstrated included bread (7 grain, white & wheat), pastries (Pate aChoux), cookies, pasta and dumplings. The resulting products performedwell in taste tests and the inclusion of strain MK7 flour was notdetectable to those tasting the products.

Example 18 MK7 Extender

Fusarium strain MK7 biomat, produced as described above, was used tocreate particles of biomass that were used as an addition to meat andfish as an extender (i.e. increase the amount of total food product bythe addition of strain MK7 to other exiting foodstuffs). Moist biomatswere steamed in a pot steamer at 97° C. for 0.5 hour, cooled to roomtemperature. Biomats where size reduced (i.e. chopping with a knife orfood processing in a food processor) to a desirable particle sizedistribution. Size reduced biomass was then added to different foodproducts to extend the amount of meat in the case of a meat extender orfish in the case of a fish extender. As an example of meat extension.10%, 20%, 30%, 40% and 50% additions of size reduced biomass were addedto hamburger meat. Size reduction of biomass was evaluated at a numberof different size distributions. Smaller particle sizes tended toproduce denser and creamier textures. Larger particles tended to produceproducts with more texture, more mouth feel and required moremastication before swallowing. The extended meat was them processed asthough no biomass was added. In the case of hamburger extension, spicesor binders can be optionally added and the extended meat was formed intoa patty or meat ball and cooked until the meat was cooked to theconsumer desired temperature. Cooking methods included stove top, oven,frying and grill. Taste tests showed that acceptable food products whereproduced at all loading levels and all size distributions of addedbiomass. Chicken and pork extensions where also tried at similar loadinglevels with similar cooking and tasting results.

Fish extension was also demonstrated at 10%, 20%, 30% and 40% loadings.Fish fillet and fish balls where produced by adding processed strain MK7at a variety of different size distributions ranging from smallparticles (less than 1.0 mm) to large particles (greater than 2 mm) withno deleterious effect on taste, color, smell or over all eatingexperience. In the case of small particle size additions, resultingfoodstuffs had a creamier texture. In the case of large particle sizeadditions, resulting foodstuffs had a firmer texture characterized bylarger particles which required more mastication before swallowing.Taste tests showed that acceptable food products where produced at alltested loading and size distribution levels.

Example 19 MK7 Jerky

Fusarium strain MK7 biomat, produced as described above, was used tocreate mycojerky, similar in appearance and taste to meat jerkies (i.e.beef jerky, buffalo jerky, pork jerky, chicken jerky, turkey jerky,etc.). Moist biomats were steamed in a pot steamer at 97° C. for 0.5hour, cooled to room temperature. Biomats where size reduced to a sizeconsistent with that normally found in jerky products. Size reducedbiomat pieces where in some cases seasoned for flavor and dehydrated ina Cuisinart dehydrator (model DHR-20) for 20-200 minutes with an averagedehydration time being 40-120 minutes. Dehydration time is a function ofthe amount of biomass loaded into the dehydrator, distribution ofbiomats in the dehydrator which impacts air flow in the dehydrator,water content of biomats (average water content approximately 75%), roomtemperature and desired water content in the final product. Watercontent post dehydration varied between 8% and 12% depending on desiredproduct characteristics. In some cases, perforating the biomass beforedehydration produced a product that tore more readily into small piecesthereby easing consumption. Perforation of the biomass was performed byusing a fork, knife or tenderizer tool which both perforated the biomassas well as disrupted the filament network such that it tore more easily.A large variety of spice mixtures (i.e. Cajun, cheese, soy, vinegar,herbs, sour cream & onion, liquid smoke, vegan meat flavors, etc.) whereevaluated. Spice mixtures were evaluated both before dehydration andpost dehydration. Those samples which were spiced before dehydrationoffered more taste and better adhered to the biomass than those whichwere treated after dehydration. The resulting jerkies all performed wellin taste tests.

Example 20 Myco-Chips

Fusarium strain MK7 biomat, produced as described above, were used tochips, similar in appearance and taste to potato chips or corn chips.Moist biomats were steamed in a pot steamer at 97° C. for 0.5 hour,cooled to room temperature. Biomats where size reduced to a sizeconsistent with that normally found in chip products as well as highlyprocessed into a paste and formed into a chip like geometry. Myco-chipswhere then put into a frying pan of hot oil (temperature app equal to380° F.) until brown. Cooking times varied as a function of biomassgeometry but cooked very fast, usually in under 15 seconds. Producedfried chips proved to be very palatable and capable of offering a widevariety of taste experiences dependent upon spices added to or coatedupon the biomass pre-frying.

TABLE 8 Nutritional data from Pleurotus eryngii Species name Pleurotuseryngii Common name King Oyster Mushroom Strain and source Blue -Mycodirect Mr. DNA results Positive ID Harvest Date Aug. 6, 2018 MediaMalt 001 mmol/L 33.1 Osmolality (mOsm) 169 C:N Ratio 19 Protein 19.38%

TABLE 9 Nutritional data from Sparassis crispa Species name Sparassiscrispa Sparassis crispa Sparassis crispa Sparassis crispa Common nameCauliflower Cauliflower Cauliflower Cauliflower Mushroom MushroomMushroom Mushroom Strain and source Amazon Mycodirect MycodirectMycodirect Mr. DNA results Positive ID Positive ID Positive ID HarvestDate Fruiting Body Jan. 22, 2019 Jan. 22, 2019 Jan. 22, 2019 Time (d) 88 8 Tray m2 0.022 0.022 0.022 Initl pH 6 6 6 End pH 4.89 5.47 4.44 Yield(g/m2) 156.7 90.67 149 Density (g/cm3) 0.0156 0.0121 0.011 TensileStrength 1960.03 242.91 1243.06 (g/cm2) Media Malt 001 Glycerol 002 Malt003 mmol/L 33.1 13.6 145.1 Osmolality (mOsm) 169 505 287 C:N Ratio 19 307.5 Protein 13.37%  35.71% 32.21% 46.24% Ash 6.54% 3.61% 3.99% 3.47%Carbohydrates 78.44%  51.16% 48.16% 37.57% Fat by AH Pending 9.52%15.60% 2.20% Total Fat as 1.65% Triglycerides Total Saturated Fatty0.36% Acids

TABLE 11 Nutritional data from Morchella esculenta Species nameMorchella esculenta Morchella esculenta Morchella esculenta Common nameYellow Morel Yellow Morel Yellow Morel Strain and source AmazonMycodirect Mycodirect Mr. DNA result Positive ID Positive ID HarvestDate Fruiting Body Dec. 28, 2018 Feb. 5, 2018 Time (d) 10 14 Tray m20.022 0.075 Initl pH 6 6 End pH 5.7 4.8 Yield (g/m2) 243.85 218.85Density (g/cm3) 0.083 0.0331 Tensile Strength 387.04 (g/cm2) Media Malt001 Malt 001 mmol/L 33.1 33.1 Osmolality (mOsm) 169 169 C:N Ratio 19 19Protein 30.37% 16.62% 26.29% Ash 6.98% Below detection 1.85%Carbohydrates 60.90% Not tested 64.47% Fat by AH 2.78% Not tested 14.07%Total Fat as 1.76% Not tested 7.68% Triglycerides Total Fatty Acids1.67% Not tested 7.05% Total Saturated Fatty 0.41% Not tested 1.32%Acids

TABLE 12 Nutritional data from Calvatia gigantea Species name “Calvatiagigantea” “Calvatia gigantea” “Calvatia gigantea” “Calvatia gigantea”Common name Giant Puffball Giant Puffball Giant Puffball Giant PuffballStrain and source Mycodirect Mycodirect Mycodirect Mycodirect Mr. DNAresult C. rosae C. rosae C. rosae C. rosae Harvest Date Aug. 9, 2018Aug. 20, 2018 Aug. 23, 2018 Aug. 23, 2018 Time (d) 5.7 6.2 9 5.8 Tray m20.022 0.25 0.022 0.25 Initl pH 6.5 6.5 6.5 6.5 End pH 5.62 6.25 5.07 Notrecorded Yield (g/m2) 71.42 111.1 178.6 108.3 Density (g/cm3) 0.07 0.0370.045 0.11 Tensile Strength (g/cm2) 314.1 264 135 281.1 Media Malt 001Malt 001 MK7-SF Malt 003 mmol/L 33.1 33.1 334 145.1 Osmolality (mOsm)169 169 1387 287 C:N Ratio 19 19 7.5 7.5 Protein 32.03% 34.89% 46.32%46.90% Ash Not tested 3.69% 7.81% 3.66% Carbohydrates Not tested 53.16%Not tested Not tested Fat by AH Not tested Below detection Not testedNot tested Total Fat as Not tested 8.27% Not tested Not testedTriglycerides Total Fatty Acids Not tested 7.91% Not tested Not testedTotal Saturated Fatty Not tested 1.66% Not tested Not tested Acids

TABLE 13 Nutritional data from Calvatia gigantea Species name “Calvatiagigantea” “Calvatia gigantea” “Calvatia gigantea” “Calvatia gigantea”“Calvatia gigantea” Common name Giant Puffball Giant Puffball GiantPuffball Giant Puffball Giant Puffball Strain and source Outgrow OutgrowOutgrow Outgrow Outgrow Mr. DNA result C. rosae C. rosae C. rosae C.rosae C. rosae Harvest Date Feb. 11, 2019 Feb. 11, 2019 Feb. 11, 2019Feb. 11, 2019 Feb. 11, 2019 Time (d) 6 6 6 6 6 Tray m2 0.022 0.022 0.0220.022 0.022 Initl pH 6 6 6 6 6 End pH 6.25 4.45 2.74 2.87 3.1 Yield(g/m2) 144.76 38.41 197.12 170.66 179.66 Density (g/cm3) 0.21 0.08 0.20.17 0.36 Tensile Strength (g/cm2) 562.96 1259.26 1559.23 833.33 1040Media MK7-102 5% Glyc MK7-102 5% Glyc MK7-102 5% Glyc MK7-102 5% GlycMK7-102 5% Glyc C:N Ratio 5 7.5 15 30 40 Protein 45.95% 49.26% 38.15%20.61% 24.30% Ash 4.65% 4.76% 6.20% 3.74% 4.61% Carbohydrates 45.77%38.90% 53.65% 70.19% 66.78% cis, cis-Poly 2.05% 2.76% 1.00% 1.07% 0.83%unsaturated FA Cis-Monounsaturated 0.51% 0.61% 0.35% 1.26% 0.73% FATotal Saturated Fatty 0.93% 1.10% 0.40% 0.92% 0.67% Acids Total Fat as3.63% 4.70% 2.00% 3.40% 2.33% Triglycerides Total Trans FA BelowDetection Below Detection Below Detection Below Detection BelowDetection isomers - GC

TABLE 14 Nutritional data from Fusarium venenatum Species name FusariumVenenatum Strain and source ATCC Mr. DNA result Positive ID Romer LabsToxicity Passed Harvest Date Jan. 16, 2019 Time (d) 5 Tray m2 0.25 InitlpH 4.5 Yield (g/m2) 66 Density (g/cm3) 0.85 Tensile Strength (g/cm2)866.2 Media MK7-102 10% Glycerol C:N Ratio 7.5 Protein 41.56% Ash 6.14%Carbohydrates 44.89% Total Fat as Triglycerides 7.43% Total Fatty AcidsBelow detection Total Saturated Fatty Acids 2.29%

TABLE 15 Nutritional data from Fusarium Venenatum Species name FusariumVenenatum Fusarium Venenatum Fusarium Venenatum Fusarium VenenatumFusarium Venenatum Common name Strain and source ATCC ATCC ATCC ATCCATCC Mr. DNA result Positive ID Positive ID Positive ID Positive ID C.Rosea Harvest Date Feb. 11, 2019 Feb. 11, 2019 Feb. 11, 2019 Feb. 11,2019 Feb. 11, 2019 Time (d) 6 6 6 6 6 Tray m2 0.022 0.022 0.022 0.0220.022 Initl pH 4.5 4.5 4.5 4.5 4.5 End pH 6.15 4.81 3.49 3.29 2.97 Yield(g/m2) 71.43 89.06 63.81 76.03 215.95 Density (g/cm3) 0.14 0.09 0.160.06 0.43 Tensile Strength 186.67 166.67 800 370.37 3151.52 (g/cm2)Media MK7-102 5% MK7-102 5% MK7-102 5% MK7-102 5% MK7-102 5% GlycerolGlycerol Glycerol Glycerol Glycerol C:N Ratio 5 7.5 15 30 40 Protein44.11% 43.14% 45.25% 48.11% 28.13% Ash 5.00% 5.20% 4.43% 5.22% 5.31%Carbohydrates 44.95% 44.77% 42.70% 42.25% 64.16% cis, cis-Poly 2.36%2.23% 2.80% 3.04% 1.05% unsaturated FA Cis-Monounsaturated 1.26% 0.48%0.80% 0.68% 0.57% FA Total Saturated Fatty 2.05% 1.00% 1.42% 1.28% 0.67%Acids Total Fat as 5.95% 3.89% 5.26% 5.24% 2.39% Triglycerides TotalTrans FA Below Detection Below Detection Below Detection Below DetectionBelow Detection isomers - GC

TABLE 16 Nutritional data from Fusarium strain MK7 Species name FusariumFusarium Fusarium Fusarium Fusarium Common name MK-7 MK-7 MK-7 MK-7 MK-7Strain and source MK-7 Stock Culture MK-7 Stock Culture MK-7 StockCulture MK-7 Stock Culture MK-7 Stock Culture Mr. DNA result Positive IDPositive ID Positive ID Positive ID Positive ID Harvest Date Feb. 11,2019 Feb. 11, 2019 Feb. 11, 2019 Feb. 11, 2019 Feb. 11, 2019 Time (d) 66 6 6 6 Tray m2 0.022 0.022 0.022 0.022 0.022 Initl pH 3.3 3.3 3.3 3.33.3 End pH 6.2 7.2 4.91 2.36 3.3 Yield (g/m2) 483.33 314.62 92.31 103.64156.09 Density (g/cm3) 0.48 0.31 0.07 0.1 0.05 Tensile Strength 333.331454.55 191.11 426.67 312.12 (g/cm2) Media MK7-102 5% MK7-102 5% MK7-1025% MK7-102 5% MK7-102 5% Glycerol Glycerol Glycerol Glycerol GlycerolC:N Ratio 5 7.5 15 30 40 Protein 53.09% 38.71% 46.67% 35.31% 40.74% Ash8.92% 7.67% 8.20% 10.00% 10.66% Carbohydrates 33.52% 50.74% 41.60%51.98% 44.95% cis, cis-Poly unsaturated 2.59% 1.60% 2.13% 1.23% 2.14% FACis-Monounsaturated 0.58% 0.43% 0.40% 0.12% 0.44% FA Total SaturatedFatty 0.94% 0.74% 0.80% 0.68% 0.89% Acids Total Fat as 4.24% 2.89% 3.53%2.35% 3.63% Triglycerides Total Trans FA Below Detection Below DetectionBelow Detection Below Detection Below Detection isomers - GC

TABLE 17 Nutritional data from Lenitnula edodes Species name Lenitnulaedodes Common name Shiitake Mushroom Strain and source Safeway HarvestDate Fruiting Body Protein 15.73% Ash 0.56% Carbohydrates 76.24% Fat byAH 5.80% Total Fat as Triglycerides 1.68% Total Fatty Acids 2.06% TotalSaturated Fatty Acids 0.46%

TABLE 18 Nutritional data from Agaricus bisporus Species name Agaricusbisporus Common name White Button Mushroom Strain and source SafewayHarvest Date Fruiting Body Protein 15.72%  Ash 5.57% Carbohydrates77.02%  Fat by AH 5.80% Total Fat as Triglycerides 1.68% Total FattyAcids Below detection Total Saturated Fatty Acids 0.46%

Example 22 Protein Content Data from Strain MK7 Biomats Grown on TwoDifferent Media

Biomats were dried in the oven for couple of days (at 99° C.) and leftin a desiccator for a few days. Samples were ground and prepared fortotal nitrogen analysis. About 5% of H₂O was estimated to be in thedried strain MK7 samples.

Total protein was calculated to be:

-   For membrane-grown strain MK7 on MK102 medium—41.2%-   For membrane-grown strain MK7 on AUM medium—43.8%

Media Characteristics (Particularly Suitable for Membrane Reactors)

Ionic strength Osmolality Carbon source Yield* Medium (mmol/L) (mOsm)(wt %) (g/m²) AUM 308.5 1902 Glycerol, 10% 359 MK7-102 296 1804Glycerol, 10% 507 Lignocellulose 1401.6 2723 LCB**, 10% 247.4 *Bestyield, strain MK7 biomat on membrane, 8.5 mL medium, 35 mm Petri dish,5-7 days **Lignoceelulosic biomass-derived from dry hay, treated withsulfuric acid

Growth Media Chemistry

AUM - artificial urine medium g/L CaC12*2H2O 0.37 MgSO4*7H2O 0.49 NaCl5.2 Na2SO4 1.41 Trisodium citrate 0.61 Li-lactate 0.1 KH2PO4 0.95 K2HPO41.2 NH4Cl 1.3 urea 10 creatinine 0.8 yeast extract (to account for traceelements and nucleic acids) 0.005 peptone, bacteriological (to accountfor amino acids) 1 FeSO4*7H2O 0.0012 Na2CO3 2.1 H2O to 1 L HCl to adjustpH 3.5

g/L Lignocellulosic biomass medium Ground hay 100 NH4NO3 10.5 Urea 3.5CaCl2 1 MgSO4*7H2O 1 KH2PO4 4 EDTA-free trace 0.4 Yeast Extract 2 H2O to1 L H2SO4 to adjust pH MK7-102 NH4NO3 10.5 Urea 3.5 CaCl2 1 MgSO4*7H2O 1KH2PO4 4 EDTA-free trace 0.4 Glycerol 10 Yeast Extract 2 H2o to 1 L HClto adjust pH

Example 23

This example shows the comparative nutritional data from two Fusariumfilamentous fungi—strain MK7 and Fusarium venenatum.

Comparative nutritional data obtained from strain MK7 and Fusariumvenenatum

Total Protein Analysis

F.V. Mat MK7 produced by Quorn Units Analyte QCB252 SB January 2019(F.V)* Percent dry Total Protein 43-52%  42% 44.0% weight Total Fat12.0% 7.4% 12.00%  Total Fiber 23.3% 25.1%  24.0% Total sugars <0.35%0.0% 0.00% Total Ash 12.4% 6.1% Total Carb 35.4% 45.0%  36.0%(calculated) Total 1.47% Nucleotides * Product includes protein fromnon-fungal sources (egg, egg whites, yeast, wheat gluten)

Branched Amino Acid Analysis

F.V. F.V. Mat mycop- F.V. pro- rotein for duced (from fish MK7 by SBGRAS food QCB25 January Quorn applic- (see Egg Units Analyte 2 2019(F.V) ation) ref) whole Percent Tryptophan* 1.07% 1.52% 1.24% 1.6% 0.94%1.60% of total protein dry weight Cystine 0.82% 0.97% 0.8% 2.21%Methionine* 1.54% 1.66% 1.59% 2.1% 1.51% 3.08% Alanine 10.43% 5.94% 6.0%5.58% Arginine 5.67% 5.94% 7.3% 4.72% 6.33% Aspartic 10.30% 10.77% 10.3%10.35% Glutamic 11.56% 11.46% 12.5% 13.17% Glycine 5.58% 5.25% 4.3%3.35% Histidine* 2.11% 2.90% 2.69% 3.5% 2.48% Isoleucine* 5.42% 4.56%3.93% 5.2% 3.96% 5.02% Leucine* 8.25% 7.04% 6.55% 8.6% 5.85% 8.56%Phenylalanine* 2.52% 4.01% 3.72% 4.9% 5.22% Proline 4.91% 5.52% 4.5%3.78% Serine 4.98% 4.97% 5.1% 7.78% Threonine* 5.99% 4.97% 4.21% 5.5%3.77% 4.39% Total lysine* 7.12% 8.98% 6.28% 8.3% 5.66% 6.88% Tyrosine2.33% 3.73% 4.0% 4.08% Valine* 9.39% 9.81% 4.14% 6.2% 4.72% 6.17%Essential AA 43.42% 45.44% 34.34% 45.9% 43.4% total Branched 23.06%21.41% 14.62% 20.0% 19.8% chain

Vitamins

F.V. Mat MK7 produced by Quorn Units Analyte QCB252 SB January 2019(F.V) IU/100 g wet Vitamin A 7.53 44 mg/100 g wet Folic acid 0.15 mg/100g wet B3 niacin 2.06 0.35 ug/100 g wet B12 1.82 mg/100 g wet B2 0.890.23 mg/100 g wet B5 0.33 0.25 mg/100 g wet B1 0.01 0.01 IU/100 g wet D2222 mg/100 g wet Omega-3 148 400 Linolenic mg/100 g wet Calcium 229 117042.5 mg/100 g wet Iron 3.88 2.8 0.5

Fatty Acid Analysis

FV mat January Units Fatty acid 2019 MK7 percent of total Capric acid(C10:0) 0.00% triglycerides Myristic acid (C14:0) 0.00% 1.91%Pentadecanoic acid 0.00% (C15:0) Palmitic acid (C16:0) 17.32% 26.75%Palmitoleic acid (C16:1) 0.00% Margaric acid (C17:0) 0.00% Stearic acid(C18:0) 11.02% 7.64% Oleic acid (C18:1 z-9) 19.69% 21.66% Linoleic acid(C18:2 44.88% 32.48% (z-9, 12) Gamma linolenic acid 0.00% Alphalinolenic acid 0.00% 3.82% Arachicid acid (C20:0) 0.00% 11-Eicosenoicacid C20:1 0.00% (z-11) Behenic acid (C22:0) 0.00% Lignoceric acid(c24:0) 0.00% Other >C20 0.00% Other <C20 0.00% 2.55% Saturated 32.80%41.40% Monounsaturated 20.00% 22.29% Polyunsaturated 47.20% 36.31%

Example 24 RNA Content Measurement from Various Filamentous Fungi

Purine Analysis Experimental Procedure: Preparation of 1000 μg/ml PurineStandards: 100 mg of each purine base (Adenine, Guanine, Xanthine andHypoxanthine) were added separately to 4 100 ml volumetric flasks. 90 mlof ultra-purified water was added to each flask and the flasks wereshaken to divide the purine solids. 10M NaOH prepared in ultra-purifiedwater was added dropwise until the purine base began to dissolve. Flaskswere shaken repeatedly until all solids were fully dissolved. Additionalbase was added as needed to provide complete dissolution. Flasks werecapped and stored under refrigeration.

Preparation of 200 μg/ml mixed purine standard: 20 ml of each 1000 ug/mlpurine standard was added to a 100 ml volumetric flask the volume wasbrought to 100 ml by addition of water. Stored under refrigeration

Preparation of pH 2.5-2.8, 150 mM Phosphate Buffer: 150 mmol sodiumphosphate buffer solution was prepared by dissolving 74.88 g of NaH2PO4in 2 liters of ultra-purified water. The solution was filtered thru a0.45 um pore size filter under vacuum. 7.88 ml of 80% phosphoric acidwas added to 2 liters of ultra-purified water and the resultingsolutions were combined. If outside the buffer range of pH 2.5-2.8 thepH can be adjusted by dropwise addition of 80% phosphoric acid to anapproximately pH 2.6 endpoint.

Sample preparation: Freeze dried mushroom samples were processed asobtained. Wet samples were transferred to tared 15m1 falcon tubes. Thesamples were then frozen at −80 F in preparation for lyophilization. Thepre-frozen samples were lyophilized for 24-48 hrs. on a Labconcolyophilizer until dry.

Dried samples were weighed, and the weight recorded. The dried materialwas ground to a fine powder in a mortar and pestle and weighed into a50m1 Erlenmeyer flask to obtain ca 500 mg sample. 15 ml of 70%perchloric acid was added to the reaction flask. The reactions were thenheated to 95C in a water bath for lhr with stirring by magnetic stirbar. The flasks were removed from the water bath and a 5 ml sample wasremoved from each reaction and transferred to a 15 ml falcon tube. pHwas increased by dropwise addition of a 10M KOH solution until thesolution was at pH4. The volume of the neutralized sample was recorded,and the solution was centrifuged at 4000 rpm for 30 min. pH was checkedafter centrifugation and adjusted as necessary, the final volume wasrecorded. 3 ml of the supernatant was drawn up with a syringe andfiltered thru a 0.45 um syringe filter for submission to HPLC analysis.

HPLC Conditions:

-   HPLC: Waters e2695 Separations Module-   Column: Shodex Asahipak GS-320HQ 7.5 mm×300 mm-   Solvent: 150 mM Sodium Phosphate buffer (pH 2.5-2.8)-   Injection volume: 10 uL-   Flow rate: 0.6 mL/min-   Column temperature: 35° C.-   Detection wavelength: 260 nm

RNA Content

RNA Species wt % in dry biomat Hericululm erinaceus 0.76-2.23 (Lion'sMane) Sparassis crispa 1.56 (cauliflower) Pleurotus ostreatus 0.95(pearl) Morchella esculenta 0.51 Morchella conica 0.14 Fusariumvenenatum 3.52-4.86 MK7 strain  1.3-1.95

Example 25 Toxicity Data from the Biomats of Filamentous Fungi Fusariumvenenatum and Morchella conica (Black Morel)

Toxicity/Growth Studies with Daphnia magna

Fusarium venenatum biomat did not result in acute toxicity to Dapniamagna, a highly sensitive macroinvertebrate commonly used for toxicityassays (EPA Publication, 1987; Guilhermino et al., 2000).

Live D. magna was purchased from Carolina Biological Supply (Cat#142330, Burlington, N.C.). Immediately after receiving the culturesfrom the supplier, 100 mL of the liquid medium containing live D. magnawas mixed with 800 mL of Arrowhead Spring Water (Nestle Waters NorthAmerica, Inc. Stamford, Conn.) in a sanitized glass bowl (rinsed with70% isopropanol and dried). The culture was gently mixed by stirringwith a sanitized plastic spoon and 400 mL of this liquid, without anyDaphnia, was removed and stored in an Arrowhead Spring water bottle at4° C. for later use. One-quarter of a pellet of Daphnia food (CarolinaBiological Supply, Cat #14-2316) was added to the remaining 500 mL ofliquid in the bowl containing the Daphnia. The bowl was covered with aloose-fitting plastic wrap and the culture was incubated at roomtemperature (21±2° C.) as directed in the manual provided by thesupplier. After 48 hours of growth and observation, the live Daphniawere used for the growth/toxicity experiments.

Sterile Petri dishes (Fisherbrand 100×15 mm, Cat #08-757-13,Thermo-Fisher) were filled with 35 mL of the liquid growth medium(mixture of spring water/Carolina Biological Supply medium) that wasstored at 4° C. (described above). The liquid medium was allowed toequilibrate to room temperature. Four active Daphnia, each approximately1-1.2 mm in length, were captured with an eye dropper (CarolinaBiological Supply, capture method described in suppliers manual) andadded to each of the eight Petri dishes. Additionally, four of the Petridishes received 0.15 g of moist F. venenatum biomat (18% solids) andfour Petri dishes received 0.03 g dry Daphnia food. The biomass wasproduced after 5 days of growth on MK7-102 medium, harvested from thetray, gently pressed by hand to remove excess medium and steamed for 30minutes to kill the cells. The steamed biomass was pressed in aFrancesco Palumbo grape press to remove liquid until a moisture contentof approximately 18% solids (82% liquid) was obtained. The pH in boththe control and F. venenatum biomass containing Petri dishes ranged from7.4 to 7.8 during the toxicity experiments. Live Daphnia were countedevery 24 h in each of the Petri dishes, as shown in the following table.

After four days, the control treatment had an average of 3.50 liveDaphnia per dish (std dev=1.29), while the F. venenatum treatment had anaverage of 3.75 live Daphnia per dish (std dev=0.50). At this time, thecontrol treatment had an average of 0.75 deaths per dish (std dev=0.96),while the F. venenatum treatment had an average of 0.25 deaths per dish(std dev=0.5).

In summary, these data indicated that steamed F. venenatum biomass doesnot result in acute toxicity to Daphnia as accessed in the describedmanner.

Daphnia Toxicity Study on Fusarium venenatum

Adult Daphnia ~3 mm length, Temperature 20.0 ± 0.5 F. Initial FinalVenenatum t = 0 day = 1 day = 2 pH pH #1 4 3 3 7.38 7.42 #2 4 4 3 #3 4 32 #4 4 3 3 mean 2.75 std dev 0.50 Initial Final Control t = 0 t = 1 t =2 pH pH #1 4 4 4 7.4 7.63 #2 4 2 1 #3 4 3 3 #4 4 4 3 mean 2.75 std dev1.26 Immature Daphnia ~1 mm length, Temperature 19.2 ± 0.5 F. venenatumInitial Final immature t = 0 day = 1 day = 2 day = 3 day = 4 pH pH #1 44 4 4 4 7.47 7.58 #2 4 4 4 4 4 #3 4 4 4 4 4 #4 4 4 4 4 3 mean 3.75 stddev 0.50 Control Initial Final Infants t = 0 t = 1 t = 2 t = 3 t = 4 pHpH #1 4 4 5 5 5 7.38 7.75 #2 4 4 4 4 4 #3 4 3 3 3 3 #4 4 2 2 2 2 mean3.50 std dev 1.29

Mycotoxin Content

Method Reference US-Multitoxin LCMSMS 45-2-LWI

Fusarium venenatum 1 Black Morel (biomat unground, (biomat unground,cooled) frozen) Alfatoxin B1 <1.3 ppb <1.3 ppb Alfatoxin B2 <1.2 ppb<1.2 ppb Alfatoxin G1 <1.1 ppb <1.1 ppb Alfatoxin G2 <1.6 ppb <1.6 ppbFumonisin B1 <0.1 ppm <0.1 ppm Fumonisin B2 <0.1 ppm <0.1 ppm FumonisinB3 <0.1 ppm <0.1 ppm Ochratoxin A <1.1 ppb <1.1 ppb Deoxynivalenol <0.6ppm <0.6 ppm Acetyldeoxynivalenol <0.8 ppm <0.8 ppm Fusarenon X <0.4 ppm<0.4 ppm Nival enol <0.6 ppm <0.6 ppm T-2 Toxin <0.2 ppm <0.2 ppm HT-2Toxin <0.2 ppm <0.2 ppm Neosolaniol <0.4 ppm <0.4 ppm Diacetoxyscirpenol<0.4 ppm <0.4 ppm zearalenone <51.7 ppb <51.7 ppb

Example 26 Characterization of a Liquid Dispersion Comprising Strain MK7Biomat Particles

Materials: MK7 vegan milk sample: app. 8.25% solid in water

All the measurements were done at room temperature (25° C.), unlessotherwise noted.

Appearance and color: Appearance is slightly off white with slight beigetones. Smell of is very slightly woody.

Refractive index, density and particle size analysis in 10-1000× dilutedsamples are illustrated in FIG. 20.

Dynamic light scattering (DLS) was used to analyze the particle size insolution.

Milk structure under optical microscope is illustrated in FIGS. 21A(10×) and 21B (100×).

Fat content of the sample (8.25%) was found to be 0.6 g/100 g.

pH content was found to be as shown below.

Concentration 0.0825% 0.825% 4.125% 8.25% pH 6.73 6.17 5.99 5.91

Viscosity: The viscosity of vegan milk sample at original concentration(8.25%) was studied. FIG. 22 was obtained by averaging three repeatingmeasurements. The sample viscosity decreases with the increasing shearrate, indicating that it is non-Newtonian fluid with sheer-thinningbehavior. It also means that the particles are much easier to getdeformed under large shear rate. For a giving shear rate, the viscosityof sample can be determined by the power-law equation. This provides theopportunity to compare this vegan milk sample to other cow's milks orsmoothies.

No visible separation of the milks was observed after 2 weeks or 4weeks.

Example 27 Process for Making a Liquid Dispersion Using the Strain MK7Filamentous Fungus

In a high speed blender (Vitamix) 200 grams strain MK7, 600 grams waterand 1 teaspoon vanilla extract were combined and blended until smooth(at least 90 seconds). The blended mixture was heated on low heat untilfungal scent was reduced, preferably to the point of being non-existent(app. 20-30 minutes). The heat was kept low to achieve a maximum of slowspeed rolling boil, which initiated when the MK7 milk reached app. 80 C.The MK7 milk was lightly boiled, temperature between 90-92 C and wasallowed to boil without disruption without stirring or whisking. Theheated blended mixture was allowed to cool and use.

After the heat cycle was completed and the milk was removed from thepot, a residual line was observed on the inside of the vessel at theintersection of the MK7 milk and air. The residue was hard and a wellattached amalgamation of dehydrated/cooked strain MK7.

Example 28 Crepes

Ingredients:

-   -   King Arthur All Purpose Flour—11.7% Gluten content.    -   C &H Granulated Sugar.    -   Strain MK7 Biomass—QCB-249-10% Glycerol—Sep. 3, 2018.    -   MK7 Milk—QCB-249-10% Glycerol—Sep. 3, 2018 (25% Biomass before        cooking)    -   Nielsen Vanilla Paste.    -   Plugra Unsalted Butter 82% Fat.

Equipment:

-   -   De Buyer Teflon Crêpe Pan.

MK7 Crêpe 1

All Purpose Flour 120 g Sea Salt 0.5 g Granulated Sugar 50 g Fresh WholeEggs 100 g Fresh Egg Yolks 30 g MK7 Milk 250 g Vanilla Paste 5 gUnsalted Butter 30 g Clarified Butter As Needed Fine Turbinado Sugar AsNeeded Total 585.5 g

The original fresh milk content of a crêpe batter was replaced with MK7milk. Milk was produced as follows: 200 grams strain MK7, 600 grams ofwater, 2 grams vanilla. Mixture was size reduced in a vitamix and heattreated without nitrogen treatment for 20 minutes at low temp boil. Thecrêpe batter was too thick.

MK7 Crêpe 2

All Purpose Flour 120 g Sea Salt 0.5 g Granulated Sugar 50 g Fresh WholeEggs 150 g Fresh Egg Yolks 30 g MK7 Milk 250 g Fresh Whole Milk 100 gWater 50 g Vanilla Paste 5 g Unsalted Butter 30 g Clarified Butter AsNeeded Fine Turbinado Sugar As Needed Total 785.5

A combination of liquids was used to adjust the batter thickness (wholeeggs, fresh milk and water). This adjustment yielded appropriate textureand flavor. Sugar may be omitted for savory crepes and savory flavorslike onion and garlic powders and herbs added.

MK7 Crêpe 3

All Purpose Flour 80 g MK7 Flour 40 g Sea Salt 0.5 g Granulated Sugar 50g Fresh Whole Eggs 150 g Fresh Egg Yolks 30 g MK7 Milk 250 g Fresh WholeMilk 100 g Water 50 g Vanilla Paste 5 g Unsalted Butter 30 g ClarifiedButter As Needed Fine Turbinado Sugar As Needed Total 785.5

Part of the flour was replaced with MK7 flour to increase the proteins.It was observed that the MK7 flour crêpe batter requires more liquidsince strain MK7 has a 150% hydration power (flour standards are 60 to70%). The texture of the crepe was more of a tortilla without theaddition of extra water.

Example 29 Pasta Dough Ingredients:

-   -   Strain MK7 Biomass—QCB-249-10% Glycerol—Sep. 3, 2018    -   MK7 Flour—QCB-249-10% Glycerol—Sep. 3, 2018    -   Bob's Red Mill Semolina.    -   Delallo 100% Organic Double 00 flour.    -   Bottled Water—PH:6.5.

Equipment:

KitchenAid food processor and pasta roller.

-   -   1. Pasta dough made with semolina, MK7 flour and water.

Ingredients Weights Semolina 100 grams MK7 Flour 50 grams Cold Water 100grams (66.66% Hydration) Sea Salt 4 grams Total 254 grams

Dough was mixed by hand and no strain MK7 taste was detected. MK7 flourincreases the need of liquid hydration from 50% in a regular pasta doughto app. 66.66% for a dough made with ⅔ semolina and ⅓ MK7 flour.

-   -   2. Pasta dough made with semolina, MK7 flour and eggs.

Ingredients Weights Semolina 100 grams MK7 Flour 50 grams Cold WholeEggs 100 grams (66.66% Hydration) Sea Salt 5 grams Total 255 grams

Dough was mixed by hand. No detection of strain MK7 taste. The egg basedough had a richer flavor. The texture was similar to trial #1 butslightly less gluten development caused by the fat in eggs (⅓ fat inyolks, so about 13% of the total mass of trial #2). The egg base doughwas harder to mix by hand because of the fat interfering with thegluten. A food processor may be used to produce texture that is smootherand like trial #1. The 20% increase of salt in the pasta dough gave goodresults.

-   -   3. MK7 Flour Hydration

Ingredients Weights MK7 Flour 50 grams Cold Water 75 grams (150%Hydration) Total 125 grams

Dough was mixed by hand. Strain MK7 needs about 150% of water to hydratein a similar manner to standard flour and starch comparative dough. Forreference: white wheat flours require 60 to 70% hydration and starches70 to 90%.

-   -   4. Pasta dough made with double 00 flour, MK7 flour and water to        compare to trial #1.

Ingredients Weights Double 00 Flour 100 grams MK7 Flour 50 grams ColdWater 100 grams (66.66% Hydration) Sea Salt 5 grams Total 255 grams

Dough was mixed by hand. No detection of strain MK7 taste. The texturewas smoother than trial #1 with the double 00 flour, which confirmed theperformance of a much finer milled MK7 flour.

-   -   5. Pasta dough made with double 00 flour, MK7 flour and eggs to        compare to trial #2.

Ingredients Weights Double 00 Flour 100 grams MK7 Flour 50 grams ColdWhole Eggs 100 grams (66.66% Hydration) Sea Salt 5 grams Total 255 grams

Dough was mixed in the food processor. No detection of strain MK7 taste.The texture was smoother than trial #2 with food processor and thenfinished by hand, which confirmed that the food processor performsbetter for fat base pasta dough.

-   -   6. Pasta dough made with semolina, double 00 flour, MK7 flour        and water to compare to trial # 1 and #4.

Ingredients Weights Semolina 50 grams Double 00 Flour 50 grams MK7 Flour50 grams Cold Water 100 grams (66.66% Hydration) Sea Salt 5 grams Total255 grams

Dough was mixed by food processor and finished by hand. No detection ofstrain MK7 taste. The texture was very similar in terms of smoothness astrial number 4. The combination of two flours with MK7 flour resulted ina diminishment of the semolina like texture and lessened the wheat flourtexture.

-   -   7. Pasta dough made with semolina, double 00 flour, MK7 flour        and eggs to compare to trial # 2 and #5.

Ingredients Weights Semolina 50 grams Double 00 Flour 50 grams MK7 Flour50 grams Cold Whole Eggs 100 grams (66.66% Hydration) Sea Salt 5 gramsTotal 255 grams

Dough mixed by food processor and finished by hand. No detection ofstrain MK7 taste. The texture was smooth, similar to trial number 5, andthe same as above with the pairing of the two flours with MK7 flour.

Example 30 Spatzle Ingredients:

-   -   MK7 Biomass—QCB-249-10% Glycerol—Sep. 3, 2018    -   MK7 Flour.    -   King Arthur All Purpose Flour 11.7% Gluten

Equipment:

-   -   None

Trial #1:

Ingredients Weights All Purpose Flour 200 grams MK7 Flour 100 gramsWhole Eggs 200 grams (66.66% Hydration) Sparkling Water 160 grams SeaSalt 8 grams Total 668 grams

Dough was mixed by hand and dispersed with a dough scraper. No detectionof strain MK7 taste. It was decided that the spatzle could be more moistinside and would benefit from an addition of cream or similar i.e. heavycream, crème fraiche, sour cream, buttermilk, yogurt, as well asaddition of Nutmeg. It was allowed to dry overnight after cooking andfinished the following day.

Trial #2:

Ingredients Weights All Purpose Flour 200 grams MK7 Flour 100 gramsWhole Eggs 200 grams (66.66% Hydration) Sparkling Water 160 grams CremeFraiche 40 grams Sea Salt 8 grams Nutmeg Powder 0.5 grams Total 708.5grams

Dough was mixed by hand. No detection of strain MK7 taste. The restedovernight spatzle had the proper color when finished in butter. Thecream addition brought the necessary moisture. Numerous chefs at theFrench Pastry School approved of the product.

Example 31 Bacon Ingredients:

-   -   Strain MK7    -   2 tablespoons Soy    -   1.5 tablespoons A1    -   1 teaspoon liquid smoke    -   2 tablespoons nutritional yeast    -   ½ teaspoon paprika    -   1 teaspoon honey

Strain MK7 biomat was cut into strips. A marinade/coating was prepared.Both sides of strain MK7 were coated with coating a and fried in oiluntil browned, patted dry, and dehydrated in dehydrator to drive offexcess moisture, until desired texture is achieved. The resulting baconanalogue performed well in terms of both texture (i.e. crispy) andflavor (had an appealing bacon like flavor). Chefs at the French PastrySchool approved of the product.

Example 32 Bread Ingredients:

-   -   0.5 cup unsweetened 7-grain cereal (store bought)    -   2 cups boiling water    -   1 envelope dry yeast    -   3 cups bread flour    -   1 cups MK7 flour    -   1 tablespoon olive oil    -   1 tablespoon dark brown sugar    -   1.5 teaspoons salt    -   2 teaspoons sesame seeds    -   2 teaspoons flax seeds    -   2 teaspoons poppy seeds    -   2 cups water

2 cups boiling water was poured over 7 grain serial and allowed to soakfor 20 minutes to soften the grain. Yeast was added to softened cereal.1 cup bread flour, oil, sugar and salt was added and stirred gentlyuntil smooth. MK7 flour was mixed with remaining bread flour and slowlymixed into above until a dough was formed. Covered and let rest (15-20minutes). Note: resting should be in a warm place. The dough was kneadeduntil smooth and elastic. Additional flour was added as required. Thekneading was for about 10-15 minutes. A large bowl was oiled. Dough wascoated with oil, placed in oiled bowl and covered. The dough was allowedto rise in warm area until doubled, about 1.5 hours. Seeds were mixed inand the dough was punched down. (Turn dough out onto lightly oiledsurface. Knead briefly with app ½ seeds.) The dough was shaped into aloaf. Baking sheet was sprinkled with app. 2 teaspoons seeds. Loaf wasplaced atop seeds, covered with towel and allowed to rise in warm areauntil almost doubled, about 30 minutes. An oven rack was positioned incenter of oven and another oven rack at the bottom of the oven. Oven waspre heated to 425 F. Loaf was brushed with water. Remaining seed mixturewas sprinkled on loaf. Diagonal slashes app. ⅛″ deep were cut on surfaceof loaf and baking sheet with loaf was placed in oven. (2 cups water waspoured into hot pan on lower rack in oven for steaming.) loaf was bakeduntil golden and crusty and tester inserted into center came out clean,about 35 minutes. The resulting bread had good crumb and very goodtaste. The addition of MK7 flour was not detectable in the resultingloaf.

Example 33 Vegan Chocolate Ice Cream Ingredients

-   -   95 grams raw Cashews    -   750 grams MK7 milk    -   0.7 grams Xanthan Gum    -   0.2 grams Instant Espresso    -   20 grams Cocoa Powder    -   1 gram Salt    -   2 grams Vanilla Extract    -   110 grams Turbinado Sugar (melted into MK7 milk)    -   150.5 grams Green & Blacks dark Chocolate 70%    -   20 grams Ghirardelli sweet cocoa powder (baking chocolate)    -   15 grams Rumford cornstarch (non-GMO)

1:3 MK7 milk analog: In a high speed blender (Vitamix) 200 grams strainMK7, 600 grams water and 1 teaspoon vanilla extract, was combined andblended until smooth (app. 90 seconds). Blended mixture was heated onlow heat for 20 minutes. (In another version of the recipe, bothblending and heat treatment was also done under nitrogen (i.e. bubblenitrogen through the MK7 milk analog during either or both the sizereduction in the Vitamix and/or the heat cycle). Use of Nitrogenresulted in an increase in milk analog creaminess and produced a sweetflavor.) Heat was kept low; to a maximum of slow speed rolling boil.Sugar was added to MK7 milk during the last 5 minutes of heat treatmentand mixed to melt into milk. (If MK7 milk is pre-made, heat milk and addsugar to melt.) A foam was visible on top with small parts of the lowerliquid showing signs of slow boiling.

In a high-speed blender, all ingredients were combined and blended onhigh for 120 seconds. According to standard teachings, it is suggestedto keep a small part of the mixture aside and dissolve the starch intothe mixture separately while whisking briskly to avoid clumping. If astarch mixture is made separately, adding it to the master mix should bedone gradually to avoid clumping.

The mixture was poured into ice cream making vessel and covered withcling film with cling film pressed against the surface of the mixture soa skin does not form while mixture is cooling. The mixture was cooledbut not frozen. The mixture was churned in ice cream maker for at least30 minutes but not more than an hour (depending on desired consistency).Packaged into appropriate container and frozen overnight.

Example 34 Chicken Nuggets Ingredients

-   -   200 g strain MK7    -   4 g chicken stock (can be meat based chicken stock or vegetarian        based chicken stock)    -   4 g binder (egg albumen or vegetarian binder)    -   3 g fat (duck fat works great as does cocoa butter. Other fats        have also been successfully used)

Biomat particle size reduction was done with a knife to achieve thedesired particle size. All ingredients were combined and processed untilthe desired particle size distribution was achieved. (If using a foodprocessor, it is recommended to pulse the food processor for 5 secondsand use a spatula to remove biomass stuck to the sides and re-integrateinto that biomass closer to the blades, and repeat this 3 times. One canprocess more or less as desired for the final product.) The size reducedbiomass was thoroughly mixed with a spoon/spatula to insure that allingredients were intimately mixed. Patties of the desired size wereformed and steamed for 30 minutes to set the binder. (Note that somebinders may require more or less time.) The steamed patties were cooled.In some patties, breading was applied and heat treated to securebreading to patty

Example 35

Burger Breakfast Sausage

Ingredients: Spice mix

-   -   30 g molasses    -   30 g tamari    -   15 g sunflower oil    -   15 g A1 sauce    -   21 g ground flax seed    -   21 g nutritional yeast    -   14 g whole wheat four    -   6 g black pepper    -   6 g sage    -   1 g thyme    -   0.5 g nutmeg

Ingredients: Burger

-   -   100 g food processed strain MK7 (Starch feedstock)    -   100 g TVP (Textured Vegetable Protein: re-constituted from dried        state in a half and half mixture of vegetable and mushroom        bullion)    -   20 g of spice mix    -   20 g cooked brown rice    -   15 g cooked onion (yellow onion cooked in oil until softened)    -   4 g dried egg albumin    -   4 g cocoa butter    -   Optional but recommended: food grade red dye (see Pic)        In a mixing bowl the spice mixture was combined and mixed until        a homogeneous mixture was obtained. This was allowed to rest for        15 minutes so that the liquids were sufficiently absorbed        producing a paste like mixture. In a food processor, the        particle size of the biomass was reduced to the desired size        i.e. a size consistent with that expected from burger (or        sausage). The burger ingredients were combined in a bowl and        mixed until homogeneous. With dampened hands, burger patties        were formed. The patties were steamed for 30 minutes or baked at        350 for 30 minutes, and fried.

Example 36 Hot Dog Bologna Ingredients

-   -   250 g strain MK7    -   1 g ground cumin    -   1 g ground cardamom    -   1 g mace    -   1.5 g black mustard seeds    -   1 g ground coriander    -   4 g black pepper    -   4 g salt    -   5 g fresh minced garlic    -   6 g granulated sugar    -   4 g paprika    -   50 g onion, peeled and chopped    -   18 g vegetable oil (app. 3 tablespoons)    -   9 g soy sauce (app. 2 tablespoons)    -   30 g almond meal    -   140 g wheat gluten    -   2 g arrowroot

All of the ingredients except the gluten and arrowroot were added to afood processor. The mixture was processed until completely smooth for atleast 60 seconds, and the processed mixture was transferred into a largebowl with a spatula. Wheat gluten and arrowroot was stirred in with awooden spoon and worked into a dough. It was noted that addition of thegluten and arrowroot formed a dense dough. The amount of gluten and theamount of processing the strain MK7 may be reduced as desired to achievea desired density.

The dough was placed on a clean surface, and divided into 8 equalportions. 8 pieces of wax paper and 8 pieces of Al foil of a sizecapable of encasing a hot dog of the appropriate size were prepared.Each portion of dough was rolled into a shape similar to that of a hotdog. on a table with a consistent pressure from your hand. If dough isdry or has cracks, it may be worked with wet hands thereby forming asmooth surface. Each hot dog was individually wrapped in wax paper andagain in aluminum foil. The ends of the foil were twisted into a tootsieroll shape. The hot dogs were steamed in a steamer for 45 minutes. Eachhot dog was unwrapped and let cool.

Example 37 Membrane Comparison

A number of simple bioreactor setups were constructed to compare theeffects on biomat growth of membrane material. Biomats were grown ineach bioreactor under identical conditions (sealed and humid, 26° C.,fungus inoculated through the growth medium) and sampled at day 6. Theresults are shown in Table 19.

TABLE 19 Comparison of membrane materials Hydro- Membrane MembraneMembrane philic or Up- Down- 1 1 pore Membrane 2 pore hydro- growth,growth, material size, μm 2 material size phobic? g/m² g/m² None n/aNone n/a n/a 182 Polyviny- 5 None n/a Hydro- 354 391 lidene philicfluoride (PVDF) Recycled 5 None n/a Hydro- 329 333 PVDF philic Polypro-5 None n/a Hydro- 265 76 pylene phobic (PP) Polypro- 10 None n/a Hydro-292 306 pylene phobic (PP) Mixed 0.45 None n/a Hydro- 31 68 cellulosephilic esters (MCE) Polyamide 0.22 None n/a Hydro- 0 (nylon) philicPolyamide 11 Polyamide 11 Hydro- 336 365 (nylon) (nylon) philic Recycled11 None n/a Hydro- 187 nylon philic PP (blue 5 None n/a Hydro- 165membrane) philic PP (blue 5 Nylon 0.2 Hydro- 0 membrane) philic PP (blueGas Hydro- 208 membrane) 5 head- 0.2 philic space + nylon PP (blue 5 MCE0.45 Hydro- 0 membrane) philic PP (blue 5 PP (blue 5 Hydro- 167membrane) mem- philic brane) Polyviny- 5 PVDF 5 Hydro- 132 lidene phobicfluoride (PVDF) Parafilm n/a None n/a n/a 0 Saran wrap n/a None n/a n/a0 P P (blue 5 PP (blue 5 Hydro- 0 mem- philic brane) PP (blue 5 PP 10Hydro- 0 membrane) philic

As shown in Table 19, a hydrophilic PVDF membrane (MilliporeSigma,Burlington, Mass.) was the best-performing membrane material under theprovided conditions, yielding the best density of both upgrowth (i.e.growth on the upper side of the membrane) and downgrowth (i.e. growth onthe lower side of the membrane) of the biomat of any membranearrangement tested. In addition to providing the best growthcharacteristics, the membrane is flexible and remained “clean,” i.e.suitable for reuse, after harvesting of the biomat; a subsequentexperiment showed that the same membrane could be reused, with nocleaning step, to achieve approximately 80% of initial efficiency.Another subsequent experiment demonstrated comparable results betweenthe 5μm PVDF membrane shown in Table 19 and a 0.2 μm PVDF membrane whenthe fungus was inoculated on a top side (i.e. opposite the feedstock) ofthe membrane. Polypropylene and nylon membranes also provided consistenthigh yields, although it was found that polypropylene membranes canbecome clogged. Low-porosity nylon membranes did not provide sufficientstructural integrity and tended to crack mid-experiment.

Example 38 “Upside-Down” Flask Bioreactor

Referring now to FIG. 25A, a “hermetic” (tightly sealed against theenvironment) embodiment of a bioreactor according to the configurationlabeled “4” in FIG. 23 is illustrated. As FIG. 25A illustrates, thebioreactor is an “upside-down” bioreactor formed by sealing together two125 mL Erlenmeyer flasks at the neck; the membrane (PVDF, pore size 5μm) is disposed within the necks of the flasks, the feedstock isdisposed in the upper flask, and the biomat is growing downwardly fromthe membrane into the gas headspace provided within the lower flask.Again, condensed water can be seen in the bottom of the lower flask.This embodiment permits continuous replenishment of the feedstock andsemi-continuous harvesting and/or sampling of the biomat. Fungus wasinoculated within the feedstock (i.e. above the membrane).

Referring now to FIG. 25B, a biomat having a thickness of approximately1 cm has been harvested as part of a second harvest from the bioreactorafter 12 days of growth (at day 20 after inoculation, with the firstharvest conducted at day 8). The biomat grew at an average rate ofapproximately 1 mm thickness per day, producing a dry yield of biomat ofup to about 100 g/m², for at least the first three weeks afterinoculation. FIG. 25C provides the dry and wet yields of the bioreactorafter 8 days (first harvest) and 12 additional days (second harvest).This bioreactor embodiment permits the production of at least about 0.7kilograms of dry biomass per square meter per week, equivalent to about0.35 kilograms of protein per square meter per week, utilizing afeedstock derived from food waste or human waste. 0.35 kilograms perweek is approximately equal to the protein requirement for a 73-kilogramhuman; thus, one square meter of membrane growth area may be sufficientto supply the protein needs of one adult human in, e.g., crewedspaceflight applications or terrestrial applications in which availablespace is limited.

After harvesting, the PVDF membrane of this Example did not retainbiomass visible to the naked eye; as illustrated in FIG. 25B, themembrane appears “clean.” However, sufficient cells remained on themembrane to effectively reinoculate the membrane for continued rapidgrowth. Other similar experiments have shown that biomats can be regrownon a previously used membrane without the need to actively re-inoculatethe membrane.

As in several other Examples, PVDF performed particularly well as amembrane material with regard to structural integrity, cleanliness,biomat production, and ease of biomat harvesting; other materials,particularly nylon, have a tendency to fail, crack, clog, or otherwisediminish in performance after only a few days, and are thereforeinferior to PVDF in their robustness and suitability for use inlong-term (e.g. 2 to 7 weeks) experiments.

Example 39 Bag Bioreactor

Two types of “bag bioreactors” were designed, fabricated, and evaluatedfor biomat growth performance. A first bag bioreactor was constructed byfashioning a bag from a Gore-Tex material, with feedstock disposedinside the bag and the inoculum of the filamentous fungus placed on theoutside of the bag. After 7 days, a biomat ranging in thickness fromabout 1 mm near a top of the bag and about 6 mm near the bottom of thebag had formed; it is believed that this difference may be attributed tovariations in the fluid pressure imparted on various portions of the bag(i.e. greater fluid pressure on the bottom of the bag than on the top).The yield of the biomat was 498 g/m² on a wet basis (estimated 124.5g/m² on a dry basis), giving a dry production rate of about 17.8g/m²/day and a carbon conversion efficiency of about 35.8%.

A second bag bioreactor was constructed by fashioning a bag from adouble layer of a PVDF membrane having a pore size of 5 μm. In thisembodiment, the filamentous fungus was inoculated through the media(i.e. from inside the bag). After 7 days, substantially all of thefeedstock had been consumed, and the carbon conversion efficiency wasestimated at 73.4%. The biomat growth process of this bag bioreactor isillustrated in FIG. 26.

Example 40 “Upside-Down” Mason Jar Bioreactor

Referring now to FIG. 27A, a “hermetic” (tightly sealed against theenvironment) embodiment of a bioreactor according to the configurationlabeled “4” in FIG. 23 is illustrated. The bioreactor was an“upside-down” bioreactor, with an upper mason jar (not pictured)disposed above a lower mason jar (FIG. 27A) and separated by a nylonmembrane having a pore size of 0.45 After 10 days of growth, a biomathaving a thickness of about 10 mm was produced. FIG. 27A illustrates thegrowth container at day 10, with feedstock on one side of the membraneand the grown biomat on the other side of the membrane. FIG. 27Billustrates the biomat harvested at day 10, and FIG. 27C illustrates athin section thereof for use in tensile strength testing (Example 41,infra).

Table 20 provides various characteristics of the biomat illustrated inFIG. 27B.

TABLE 20 Characteristics of “upside down” mason jar bioreactor-grownbiomat Area of biomat 25.5 cm² Wet biomass 17.5 g Dry biomass 3.13 gYield (dry weight) 1226 g/m² Wet density 0.684 g/cm³ Dry density 0.123g/cm³ Fumonisins 0.0 ppm Carbon conversion efficiency 62.6%

Example 41 Tensile Strength Testing of “Upside Down” Mason JarBioreactor-Grown Biomat

A 5 cm×1 cm section of biomat illustrated in FIG. 27B was separated witha razor blade, as illustrated in FIG. 27C. The thickness of the sectionwas recorded with a caliper, and the cut sample was placed between twoglass slides such that 1 cm of the 5 cm length of the sample was pressedbetween the two slides, while care was taken not to damage the sample byapplying excessive pressure via the slides. The glass slides were thenattached to a tensile strength testing apparatus via a clip, such thatthe tension of the clip prevented the sample from slipping. Twoadditional glass slides were used to press another 1 cm length of thesample at the opposite end. A water receptacle adapted for use with thetensile strength testing apparatus was attached to the slides and filledwith water at a rate of 1 mL per second until sample failure. Uponfailure, the mass of the water, the receptacle, the clip, and theremaining sample were weighed together; by dividing this weight by thethickness of the sample, the tensile strength of the sample wascalculated. The tensile strength of the sample of biomat produced inExample 40, as determined by this method, was 1784 grams-force persquare centimeter.

Example 42 Use of “biomembrane”

Referring now to FIGS. 28A and 28B, a typical bioreactor setup isillustrated at 3 days (FIG. 28A) and 6 days (FIG. 28B) afterinoculation. Fungus was inoculated on the side of the nylon membrane(pore size 0.2 μm) opposite the feedstock, and biomats grew at a rate ofapproximately 1 mm per day. After an initial harvest at day 3, at whichtime it was observed that the nylon membrane was significantly deformed,the same membrane was reused and a biomat of approximately the samethickness grew over the following three days. At 6 days afterinoculation, it was discovered that the nylon membrane had failed, but acombination of the nylon and the biomat itself continued to act as themembrane. Thus, it has been unexpectedly found that even when theinitially provided membrane fails, the biomat itself can act as a“biomembrane” in the practice of the bioreactor.

Example 43 Human Waste Products as Feedstock/Growth Medium

One advantage of bioreactors of the present disclosure and methods ofuse thereof is that animal waste products, including but not limited tohuman urine and human feces, agricultural waste products and industrialwaste products may be used as a feedstock or growth medium. For thisExample, artificial urine medium (AUM) and artificial feces medium (AFM)were prepared according to the compositions shown in Tables 21 and 22.

TABLE 21 AUM composition Component Concentration (g/L) Calcium chloridedihydrate 0.37 Magnesium sulfate heptahydrate 0.49 Sodium chloride 5.2Sodium sulfate decahydrate 3.2 Citric acid 0.4 Lactic acid 0.1Monopotassium phosphate 0.95 Dipotassium phosphate 1.2 Ammonium chloride1.3 Urea 10 Creatinine 0.8 Yeast extract 0.005 Bacteriological peptone 1Iron sulfate heptahydrate 0.0012 Sodium carbonate 2.1 Water balance

TABLE 22 AFM composition Component Mass fraction Water 0.8 Dry baker'syeast 0.06 Microcrystalline cellulose 0.03 Psyllium 0.035 Miso paste0.035 Oleic acid 0.04 Sodium chloride 0.004 Potassium chloride 0.004Calcium chloride 0.002

Biomats were grown on various membranes in simple bioreactors, using theprepared AUM and AFM as feedstocks, in addition to samples usinglignocellulosic biomass (an efficient carbon source) as a feedstock. Thebioreactor yields resulting from these feedstock/membrane combinationsare given in Table 23.

TABLE 23 Bioreactor yields on AUM, AFM, and lignocellulosic biomassMembrane Bioreactor Bioreactor Bioreactor yield, material and yield,AFM:AUM yield, AUM lignocellulosic pore size feedstock (g/m²) feedstock(g/m²) biomass (g/m²) None 317 342 PVDF, 5 μm 274 359 247 Nylon, 0.2 μm196 145 MCE, 0.2 μm 298 MCE, 0.45 μm 221 PVDF, 0.2 μm 251 348

Biomats produced in these tests are illustrated in FIGS. 30A and 30B.Additionally, it was observed that biomats grown on 1:1, AFM,AUM adheredvery loosely to the PVDF membranes and would simply fall off when acertain thickness/weight was attained. Thus, a self-harvesting systemwas envisioned where biomats would fall away from the membranes after acertain weight was attained—no need for physical/manual removal from themembrane would be necessary

Example 44 Continuous Feeding of Feedstock in Absence of Backpressure

A bioreactor enabling continuous feeding of the feedstock to thebioreactor was constructed that eliminated the backpressure caused bydepletion of the liquid feedstock and enabled equilibration of theliquid feedstock medium with the outside environment via a gas-permeablefilter; this was accomplished by drilling a hole in the top Erlenmeyerflask and placing an air filter in the hole created thereby. Thisembodiment is illustrated in FIG. 31A, and a more generalized bioreactorsetup is illustrated in FIG. 31B.

Use of a nylon membrane having a pore size of 0.2 μm proved ineffectivein this embodiment because the membrane tended to leak. By contrast, apolypropylene membrane having a pore size of 10 μm tolerated thepressure inside the bioreactor and did not fail, crack, or leak.

Example 45 Membrane-Bag Biofilm Reactor (MBBR) System

A membrane-bag biofilm reactor (MBBR) system may provide a scalable andconvenient means for producing dense usable biomass, including by usingwastes and carbon substrates that are anticipated to be available, e.g.,on crewed space missions or in terrestrial applications. The MBBR mayprovide a lightweight, compact, simple, and reusable system forculturing filamentous fungi, which may be used, by way of non-limitingexample, not only as a foodstuff but in pharmaceuticals, nutraceuticals,fuels, leather analogues, textiles, and/or building materials.

A three-layer Gore-Tex fabric was chosen as the MBBR membrane for itsdurability and water-resistant and gas exchange properties. The bagswere fabricated by using a heat-fluxable sealing tape to providewatertight seams, and a simple roll closure system for sealing the topof the bag and attaching it to a support was employed. The outsidesurface of the bags were inoculated with filamentous fungus strain MK7and filled with a growth medium, then the tops of the bags were sealedand the bags were suspended on a rack in a sealed, temperature- andhumidity-controlled box (50 cm×50 cm×70 cm, 25±1° C., approximately 95%relative humidity). After 3 days, each bag was fully covered with alayer of strain MK7 biomat displaying a thick layer of hyphae, aerialhyphae, and mycelia. Biomat growth after 5 days is illustrated in FIG.32. By the end of the 7-day growth period, the bags were completelydepleted of the glycerol feedstock, and the calculated conversion of theglycerol feedstock into dry biomat was approximately 35%, not accountingfor any glycerol sorbed by the membrane fabric.

Example 46 Effect of Membrane Material and Pore Size on Biomat Growth

Tests in simple bioreactors were performed under identical conditionsusing four different membrane materials at five different pore sizes (20different membranes total). The characteristics of the membranes aregiven in Table 24.

TABLE 24 Membrane materials and characteristics Water Hydro- flow philicor Pore Thick- rate per Bubble Speci- hydro- size, ness, area, point,men # Material phobic? μm mm cm/min kPa 1 PTFE Hydro- 0.2 0.2 ±0.1 >6.2 >140 2 w/ phobic 0.45 >30.9 >70 3 PP 1 >86.6 >24 4 backing3 >98.17 >14 5 5 >196.9 >7 6 PP, Hydro- 0.2 >18 >14 7 no phobic0.45 >54.2 >9 8 backing 1 >120 >4 9 3 >180.5 >3.5 10 5 >240.7 >3 11Nylon Hydro- 0.2 0.085 − >4 310−410 12 w/ philic 0.45 0.14 >16 150−25013 PET 1 >85 80−120 14 substrate 3 >165 40−60 15 5 >240 30−50 16 PVDFHydro- 0.2 0.085 − >5 70−150 17 w/ phobic 0.45 0.12 >10 40−80 18 PET1 >20 25−35 19 substrate 3 >40 15−20 20 5 >50 10−15

Selected characteristics of the biomats grown on these membranes aregiven in Table 25.

TABLE 25 Biomat characteristics Specimen Thickness, cm Area, cm² Wetdensity, g/cm³ Dry yield, g/cm² # AUM MK AUM MK AUM MK AUM MK 1 0.1 0.24.9 3.14 1.05 0.71 84 261 2 0.2 0.2 3.14 9.62 0.96 0.43 169 128 3 0.250.1 4.9 9.62 0.59 0.62 245 113 4 0.3 0.4 4.9 9.62 0.75 0.31 241 195 50.2 0.5 3.94 9.62 1.17 0.28 203 165 6 0.2 0.1 4.52 9.62 1.03 0.56 55 4 70.3 9.62 0.30 37 8 0.3 9.62 9.62 0.22 109 61 9 0.2 9.62 9.62 0.48 122 7510 0.3 0.6 9.62 9.62 0.34 0.36 79 213 11 0.5 0.7 9.62 9.62 0.404 0.611342 507 12 0.2 0.55 9.62 9.62 0.412 0.380 150 298 13 0.55 9.62 0.618 53214 0.55 9.62 0.553 435 15 0.4 0.1 9.62 9.62 0.368 0.466 321 67

The minimum biomat densities observed in these experiments was 0.22g/cm³ wet and 0.036 g/cm³ dry.

Visual inspection of the specimens of this Example provided severalinsights. In PTFE and PP membranes, which were characterized by greaterthickness and tortuosity, it appeared that significant quantities ofbiomass accumulated inside the membrane itself and were thus unavailablefor harvesting; moreover, in PTFE membranes, the fungal inoculum did not“spread” to cover the entire surface of the membrane but had to beapplied via a spray, a vacuum pump, a Q-tip, a paintbrush, or the like,and biomat did not grow across the entire membrane area as a result.Additionally, the PET-backed nylon membrane did not suffer the samestructural integrity problems (cracking, failure, etc.) evidenced by thepure nylon membranes of earlier Examples. It is hypothesized that thepoor results for hydrophobic PVDF with PET backed membranes was causedby the thinner, smoother membrane used for these experiments, which mayhave resulted in less ability for fungal bodies to get purchase on themembrane (a problem which could potentially be solved by roughening themembrane), but PVDF membranes also were characterized by the formationof a thick “slime” that requires further study. Another possibility isthat hydrophobic PVDF with PET backing performance was more due tosignificantly lower liquid flow rates through the membranes, compared tothe same thin and smooth nylon membrane (see table 24). Nylon and PVDFmembranes also came out quite “clean,” i.e. suitable for reuse. 0.2hydrophobic PVDF-PET was quite clean, but 0.45, 1, 3 or 5 had slime.Hydrophylic PVDF 0.2 or 5.0 did not have a slime (see data in table 19).

One further result of note is that while yields tended to increase withincreasing pore size for PTFE and PP membranes, the opposite trend wasobserved for PET-backed nylon membranes. Without wishing to be bound byany particular theory, it is hypothesized that the hydrophilic nature ofthis membrane might, in large-pore embodiments, have provided conditionstoo “wet” for optimal biomat growth, and that the trend may be differentif the membrane were “flipped” to attempt to grow the biomat on thehydrophobic surface instead.

Example 47 Tensile Strength Testing

The tensile strength of various biomat samples was tested according tothe procedure described in Example 41. Biomat characteristics andtesting results are given in Table 26.

TABLE 26 Biomat characteristics Membrane Biomat Biomat Wet Test Tensilearea, age, thickness, density, strip strength, Membrane Medium cm² daysmm g/cm³ area g_(f)/cm² Nylon, MK 25.5 10 10 0.68 4 cm × 1784 0.2 μm 0.1cm PVDF-PET, AUM 25.5 10 5 0.52 2 cm × 998 0.2 μm 1 cm (hydrophobic)PVDF, AUM 9.62 7 5 0.37 2.6 cm × 1080 0.2 μm 0.9 cm (hydrophilic)Nylon-PET, MK 9.62 5 5.5 0.62 2.4 cm × 661 1.0 μm 0.8 cm Nylon-PET, MK9.62 5 7 0.61 2.4 cm × 682 0.2 μm 0.9 cm Nylon-PET, AUM 9.62 5 5 0.402.3 cm × 748 0.2 μm 0.9 cm

Example 48 Backpressure-Eliminating Bioreactors

Bioreactors that eliminate backpressure resulting from consumption ofthe feedstock, substantially conforming to the generalized schematicpresented in FIG. 31A, were constructed and used to test the performanceunder fluid pressure of various membranes. Results of these tests aregiven in Table 27.

TABLE 27 Fluid pressure performance of various membrane types MembraneBacking hydro- hydro- Height philic philic of Pore or or liquid Membranesize, hydro- Backing hydro- column, material μm phobic? material phobic?cm Result Nylon 0.2 Hydrophilic None n/a n/a Immediate leaking. Nylon0.2 Hydrophilic PET Hydro- n/a Immediate phobic leaking. Polypro- 10.0Hydrophobic None n/a 11 Slow leaking, pylene with mat growth onmembrane. PTFE 0.22 Hydrophobic PP Hydro- 8.3 No leaking, phobic withmat growth on membrane. PTFE 0.45 Hydrophobic PP Hydro- 8.3 No leaking.phobic PTFE 1.0 Hydrophobic PP Hydro- 9 No leaking, phobic with matgrowth on membrane. PTFE 3.0 Hydrophobic PP Hydro- n/a Immediate phobicleaking. PTFE 5.0 Hydrophobic PP Hydro- n/a Immediate phobic leaking.

These results tend to indicate that hydrophilic membranes and membraneswith larger pore sizes are less capable of withstanding hydrostaticpressure than small-pore hydrophobic membranes. It is hypothesized,without wishing to be bound by any particular theory, that hydrophilicmembranes and large-pore membranes, which readily allow feedstock topass through, may be more suitable for applications in which thefeedstock is disposed below the membrane, whereas small-pore hydrophobicmembranes may be more suitable for applications in which the feedstockis disposed above the membrane. It is also hypothesized that variationsin fluid pressure may allow an operator of the bioreactor to adjust ortune biomat growth rate or other biomat characteristics, and/orfacilitate in situ removal of the biomat from the membrane.

Example 49 Effect of Light Conditions on Biomat Growth

Fungal strain MK7 was inoculated onto nylon and polypropylene membranesand grown into biomats in light and dark conditions. Although all matsappeared identical after 4 days of growth, by day 7 biomats grown underlight conditions had begun to die, likely due to nutrient depletion inthe feedstock. Biomats grown in dark conditions, by contrast, appearedhealthy well after day 7, and appeared to evolve small bubbles of gas,suggesting a different metabolic pathway than that of biomats grown inlight conditions. Biomats grown in dark conditions did not begin todarken and appear unhealthy until day 14. These results are illustratedin FIG. 34 (“N” denotes nylon membrane, “P” denotes polypropylenemembrane.

Example 50 Comparative Protein Content

Each of several filamentous fungi were grown by at least one methodselected from surface fermentation according to the present invention,submerged fermentation according to the methods of the prior art, andfruiting body (i.e. “natural”) growth. In each case of surfacefermentation, the carbon-to-nitrogen ratio of the liquid growth mediumwas 7.5. Average protein contents for each fungus grown by each methodare presented in Table 28.

TABLE 28 Comparative protein content Protein content Fungus Growthmethod Growth medium (wt %) Strain MK7 Surface MK7-102 51.0 R1 fructose51.4 Submerged MK7 glycerol 34.6 Sparassis crispa Surface Malt 001 46.2Fruiting body n/a 8.9 Morchella conica Surface Malt 001 44.1 Fruitingbody n/a 34.3

The results of this Example demonstrate that the surface fermentationmethods of the present invention produce filamentous fungus biomatshaving greater protein content than can be achieved by either submergedfermentation or natural growth of the fungal fruiting bodies. Even moreparticularly, the methods of the present invention produce biomatshaving protein contents of at least about 40 wt %, which for manyspecies of filamentous fungus cannot be achieved by any previously knownmethod.

Example 51 Mesh Solid Support

1.75 kilograms of a 1:1 sucrose:fructose carbon source in M2 media waspoured into a 10″×13″ inch Pyrex tray. A 0.09 m² section of a #7polyolefin mesh (mesh size 2 mm, material believed to be LDPE) wascoated with an inoculum of strain MK7 and applied to the surface of thegrowth medium, whereupon the tray was cultured at 27° C. for 72 hours.Initial biomat formation was observed to be more rapid on the meshsurface than on the bulk medium in the tray.

A first section of biomat was harvested from the tray by cutting aroundthe perimeter of the polyolefin mesh, and a second section of the biomatwas harvested from the tray by cutting around the periphery of the tray.It was immediately apparent that biomat harvested from the surface ofthe mesh (wet mass 70 g) had a much lower content of entrained liquidgrowth medium than the sample from the surface of the medium (wet mass144 g).

Both biomat samples were processed by steaming for 30 minutes, followedby soaking in water at 50° C. for 15 minutes and hand pressing. Thefinal (dried) mass of the biomat harvested from the mesh surface was 30g (333.33 g/m²), while the final (dried) mass of the biomat harvestedfrom the bulk medium was 24 g (266.67 g/m²). This Example thus providesevidence that yields of biomat can be improved by approximately 20%simply by providing a solid mesh as a scaffold or substrate to whichbiomat structures can attach.

Example 52 Refreshed Medium Experiment with King Oyster and ReishiMushroom

The experimental procedure (including control comparison) of Example 2was repeated, except that king oyster mushroom (Pleurotys eryngii) andreishi mushroom (Ganoderma lucidum) were used in place of strain MK7, amesh support as in Example 51 was used, and the experiment was allowedto proceed for a period of 22 days, with measurements of total biomatgrowth obtained at 5, 10, and 22 days. The results are given in Table 29(all yields are given in grams dry mass per square meter).

TABLE 29 Mushroom growth on mesh support vs. control King oyster ReishiDay Refresh Control Refresh Control 5 120 78 108 87 10 238 143 239 12922 444 128 433 132

Example 53 Medium Characteristics and Effect on Protein Content

An artificial urine medium (“AUM”), a lignocellulosic biomass medium(“LCBM”), and an MK102 medium were prepared according to thecompositions in Tables 28, 29, and 30, respectively.

TABLE 30 Artificial urine medium composition Component Concentration(g/L) Calcium chloride dihydrate 0.37 Magnesium sulfate heptahydrate0.49 Sodium chloride 5.2 Sodium sulfate 1.41 Trisodium citrate 0.61Lithium lactate 0.1 Bipotassium phosphate 0.95 Monopotassium phosphate1.2 Ammonium chloride 1.3 Urea 10 Creatinine 0.8 Yeast extract 0.005Bacteriological peptone 1 Iron sulfate heptahydrate 2.1 Water balance

TABLE 31 Lignocellulosic biomass composition Component Concentration(g/L) Ground hay 100 Ammonium nitrate 10.5 Urea 3.5 Calcium chloride 1Magnesium sulfate heptahydrate 1 Bipotassium phosphate 4 EDTA-free trace0.4 Yeast extract 2 Water balance

TABLE 32 MK102 medium composition Component Concentration (g/L) Ammoniumnitrate 10.5 Urea 3.5 Calcium chloride 1 Magnesium sulfate heptahydrate1 Bipotassium phosphate 4 EDTA-free trace 0.4 Glycerol 10 Yeast extract2 Water balance

The media were then acidified with hydrochloric (for AUM and MK102) orsulfuric (for LCBM) acid to achieve a pH of 3.5. The ionic strength andosmolality of the media were determined, and mats were grown on eachmedium and then desiccated and/or dried and assayed to determine proteincontent. Results of these determinations are given in Table 31.

TABLE 31 Comparison of media and mats grown therefrom Ionic strengthOsmolality Best yield Component (mmol/L) (mOsm) (g/m²) Protein contentAUM 308.5 1902 359 43.8% MK102 296 1804 507 41.2% LCBM 1401.6 2723 247.4Not reported

1-51. (canceled)
 52. A bioreactor, comprising: a container; at least onemembrane disposed within or on a surface of the container, the at leastone membrane comprising a first surface and a second surface; afeedstock for the growth of a filamentous fungus, contacting the firstsurface of the at least one membrane; and a filamentous fungus inoculum,disposed on either the first surface or the second surface of the atleast one membrane, wherein, upon culturing the inoculum in thebioreactor, a biomat of the filamentous fungus forms on the secondsurface of the at least one membrane after a biomat growth period. 53.The bioreactor of claim 52, wherein the container is a bag, wherein thefirst and second surfaces of the at least one membrane are first andsecond surfaces of at least a portion of the bag.
 54. The bioreactor ofclaim 52, wherein the feedstock is subjected to a positive or negativepressure imparted on a side of the feedstock opposite at least one ofthe first surface and the second surface of the at least one membrane.55. The bioreactor of claim 52, further comprising cyanobacteria,wherein the cyanobacteria provide at least one of oxygen gas and carbonto promote the growth of the biomat.
 56. The bioreactor of claim 52,wherein at least one of the following is true: i) a density of thebiomat is at least about 0.05 grams per cubic centimeter; and ii) adensity of the biomat after drying is at least about 0.01 grams percubic centimeter.
 57. The bioreactor of claim 52, wherein the biomatcomprises at least one layer.
 58. The bioreactor of claim 52, whereinthe biomat has a tensile strength of at least about 3 kilopascals or atleast about 30 grams-force per square centimeter.
 59. The bioreactor ofclaim 58, wherein the biomat has a tensile strength of at least about100 kilopascals or at least about 1,020 grams-force per squarecentimeter.
 60. The bioreactor of claim 52, wherein the at least onemembrane comprises at least one polymer selected from the groupconsisting of polypropylenes, polytetrafluoroethylenes, polycarbonates,polyamides, cellulose acetate, polyvinylidene fluorides, mixed celluloseesters, polyethersulfones, polyethylenes, and polypyrroles.
 61. Thebioreactor of claim 52, wherein the at least one membrane comprises atleast one material selected from the group consisting of polypropylenefabrics, polytetrafluoroethylene fabrics, and a nylon net filter. 62.The bioreactor of claim 52, wherein the at least one membrane comprisesat least one of a glass fiber material and a porous ceramic material.63. The bioreactor of claim 52, wherein an average pore size of the atleast one membrane is between about 0.2 μm and about 25 μm.
 64. Thebioreactor of claim 63, wherein an average pore size of the at least onemembrane is between about 5 μm and about 11 μm.
 65. The bioreactor ofclaim 52, wherein the container is enclosed and substantially airtight,wherein the container encloses a gas headspace into which the biomatgrows.
 66. The bioreactor of claim 52, wherein the biomat separates fromthe at least one membrane spontaneously.
 67. The bioreactor of claim 52,wherein, when the biomat is removed from the at least one membrane, anew inoculum of filamentous fungi remains on the at least one membrane.68. The bioreactor of claim 52, wherein the filamentous fungus belongsto an order selected from the group consisting of Mucorales,Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales andHypocreales.
 69. The bioreactor of claim 52, wherein the filamentousfungus belongs to a family selected from the group consisting ofMucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae,Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae,Physalacriaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae,Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae. 70.The bioreactor of claim 52, wherein the filamentous fungus is selectedfrom the group consisting of strain Rhizopus oligosporus, Ustilagoesculenta, Hericululm erinaceus, Polyporous squamosus, Grifola frondosa,Hypsizygus marmoreus, Hypsizygus ulmarius (elm oyster), Calocybegambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus,Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii,Pleurotus ostreatus (pearl), Pleurotus ostreatus var. columbines (Blueoyster), Tuber borchii, Morchella esculenta, Morchella conica, Morchellaimportuna, Sparassis crispa (cauliflower), Fusarium venenatum, strainMK7 (ATCC Accession Deposit No. PTA-10698), Disciotis venosa, andCordyceps militaris. Trametes versicolor, Ganoderma lucidum, Flammulinavelutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus,Leucoagaricus holosericeus, Calvatia fragilis, Handkea utriformis, andPholiota adiposa.
 71. The bioreactor of claim 52, wherein the feedstockcomprises at least one of feces of an animal and urine of an animal. 72.The bioreactor of claim 71, wherein the animal is a human.
 73. Thebioreactor of claim 52, wherein the at least one membrane is a singlecomposite membrane, wherein the first surface comprises a first materialand the second surface comprises a second material.
 74. The bioreactorof claim 52, wherein the at least one membrane comprises at least afirst membrane and a second membrane, wherein the first surface is asurface of the first membrane and the second surface is a surface of thesecond membrane.
 75. The bioreactor of claim 74, wherein the first andsecond membranes are in physical contact with each other.
 76. Thebioreactor of claim 52, further comprising a selective gas-permeablemembrane, wherein a first gas produced during growth of the biomat isselectively separated into a gas headspace on a first side of theselective gas-permeable membrane.
 77. The bioreactor of claim 76,wherein a second gas produced during growth of the biomat is selectivelyseparated into a gas headspace on a second side of the membrane.
 78. Amethod for producing a biomat of a filamentous fungus, comprising:inoculating a filamentous fungus in a bioreactor, wherein the bioreactorcomprises: a container; at least one membrane disposed within or on asurface of the container, the at least one membrane comprising a firstsurface and a second surface, wherein either or both of the first andsecond surfaces are adapted to receive thereon the inoculum of thefilamentous fungus; and a feedstock for the growth of a filamentousfungus, contacting the first surface of the at least one membrane.79-104. (canceled)
 105. A method for producing fresh water, comprising:inoculating a filamentous fungus in a bioreactor, wherein the bioreactorcomprises: a container; and a feedstock for the growth of a filamentousfungus; culturing the filamentous fungus to form a biomat on at leastone of a surface of the feedstock and a surface of a membrane of thebioreactor, wherein the filamentous fungus produces water as a metabolicbyproduct during growth of the biomat; and collecting water produced bythe growth of the biomat. 106-110. (canceled)
 111. A method forproducing a gas, comprising: inoculating a filamentous fungus in abioreactor, wherein the bioreactor comprises: a container; and afeedstock for the growth of a filamentous fungus; culturing thefilamentous fungus to form a biomat on at least one of a surface of thefeedstock and a surface of a membrane of the bioreactor, wherein thefilamentous fungus produces the gas as a metabolic byproduct duringgrowth of the biomat; and collecting the gas produced by the growth ofthe biomat.
 112. (canceled)
 113. A method for producing a biomat of afilamentous fungus, comprising: (a) inoculating an effective amount ofcells of at least one filamentous fungus to a first aliquot of growthmedium to produce an inoculated growth medium; (b) incubating theinoculated growth medium for a first time to produce an initial biomat;(c) removing at least a portion of the first aliquot of growth mediumand adding a second aliquot of growth medium to provide a refreshedgrowth medium; and (d) incubating the refreshed growth medium for asecond time to produce a finished biomat, wherein at least one of athickness and a dry-mass density of the finished biomat is greater thanthat of the initial biomat. 114-123. (canceled)
 124. A biomat of atleast one filamentous fungus, having a dry-mass density of at leastabout 75 grams per liter. 125-134. (canceled)
 135. A method forproducing a biomat of a filamentous fungus, comprising: inoculating afilamentous fungus in a bioreactor, wherein the bioreactor comprises: acontainer; at least one mesh scaffold disposed within or on a surface ofthe container, the at least one mesh scaffold comprising a first surfaceand a second surface, wherein either or both of the first and secondsurfaces are adapted to receive thereon the inoculum of the filamentousfungus; and a feedstock for the growth of a filamentous fungus,contacting the first surface of the mesh scaffold. 136-141. (canceled)